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United States Patent |
5,763,570
|
Kauvar
,   et al.
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June 9, 1998
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Glutathione analogs and paralog panels comprising glutathione mimics
Abstract
Compounds of the formula
##STR1##
or the alkyl (1-6C), alkenyl (1-6C), or arylalkyl (7-12C) amides or salts
including the cycloamido forms thereof;
wherein n is 1 or 2;
wherein when n is 1, X is a mono- or disubstituted or unsubstituted
hydrocarbyl (1-20C) moiety optionally containing 1 or 2 nonadjacent
heteroatoms (O, S or N), and wherein said substitution is selected from
the group consisting of halo, OR, and SR, wherein R is H or lower alkyl
(1-4C); when n is 2, one X is as above defined and the other X is lower
alkyl (1-4C);
Y is selected from the group consisting of
##STR2##
wherein m is 1 or 2; and AA.sub.C is an amino acid coupled through a
peptide bond to the remainder of the compound of formula 1, are useful as
affinity ligands, elution reagents, solution inhibitors, diagnostic
reagents and therapeutics. These compounds and analogous tripeptide
glutathione analogs can be used as members of panels to obtain specific
characteristic profiles for various glutathione-S-transferases.
Inventors:
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Kauvar; Lawrence M. (San Francisco, CA);
Lyttle; Matthew H. (Point Reyes Station, CA)
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Assignee:
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Terrapin Technologies, Inc. (South San Francisco, CA)
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Appl. No.:
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473397 |
Filed:
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June 7, 1995 |
Current U.S. Class: |
530/331 |
Intern'l Class: |
C07K 005/08; A61K 038/06 |
Field of Search: |
530/331
514/18
|
References Cited
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|
Primary Examiner: Tsang; Cecilia J.
Assistant Examiner: Borin; Michael
Attorney, Agent or Firm: Morrison & Foerster LLP
Parent Case Text
This application is a continuation of U.S. application Ser. No. 08/126,229,
filed 24 Sep. 1993, now U.S. Pat. No. 5,599,903; which is a
continuation-in-part of U.S. application Ser. No. 07/863,564, filed 3 Apr.
1992, now abandoned; which is a continuation-in-part of U.S. application
Ser. No. 07/693,245, filed 29 Apr. 1991, now abandoned. This application
is also a continuation-in-part of U.S. application Ser. No. 07/678,849,
filed 2 Apr. 1991, now U.S. Pat. No. 5,338,659; and of U.S. application
Ser. No. 07/607,875, filed 1 Nov. 1990, now abandoned; which is a
continuation-in-part of application Ser. No. 07/429,721 filed 31 Oct.
1989, now U.S. Pat. No. 5,133,866; which is a continuation-in-part of U.S.
application Ser. No. 07/355,042, filed 16 May 1989, now U.S. Pat. No.
4,963,263; and is also a continuation in part of U.S. application Ser. No.
07/447,009, filed 6 Dec. 1989, now U.S. Pat. No. 5,300,425; which is a
continuation-in-part of U.S. application Ser. No. 07/255,906, filed 11
Oct. 1988, now U.S. Pat. No. 5,217,869; which is a continuation of U.S.
application Ser. No. 07/108,130, filed 13 Oct. 1987, now abandoned.
Claims
We claim:
1. The alkyl (1-10C), alkenyl (1-10C), or arylalkyl (7-12C) esters or
amides of a compound of the formula:
##STR6##
wherein Z is selected from the group consisting of S, O and CH.sub.2 ;
wherein n is 1;
wherein X is a mono- or disubstituted or unsubstituted hydrocarbon radical
(1-20C) optionally containing 1 or 2 nonadjacent heteroatoms (O, S or N),
and wherein said substitution is selected from the group consisting of
halo, --NO, --NO.sub.2, --NR.sub.2, OR, and SR, wherein R is H or lower
alkyl (1-4C);
Y--CO is .gamma.-glu; and
AA.sub.C is phenylGly coupled through a peptide bond to the remainder of
the compound.
2. The ester or amide of claim 1 wherein X is methyl, propyl, hexyl, octyl,
benzyl, naphthyl or trityl.
3. A panel comprising at least five diverse tripeptide glutathione analogs
of the formula:
##STR7##
or the alkyl (1-10C), alkenyl (1-10C), or arylalkyl (7-12C) esters or
amides or salts including the cycloamido forms thereof;
wherein Z is selected from the group consisting of S, O and CH.sub.2 ;
wherein n is 1 or 2;
wherein when Z is O or CH.sub.2 or is S and n is 1, X is H, a mono- or
disubstituted or unsubstituted hydrocarbon radical (1-20C) optionally
containing 1 or 2 nonadjacent heteroatoms (O, S or N), and wherein said
substitution is selected from the group consisting of halo, --NO,
--NO.sub.2, --NR.sub.2, OR, and SR, wherein R is H or lower alkyl (1-4C);
when Z is S and n is 2, one X is as above defined (except H), and the
other X is lower alkyl (1-4C);
Y is selected from the group consisting of
##STR8##
wherein m is 1 or 2; and AA.sub.C is selected from the group consisting
of glycine, valine, alanine, .beta.-alanine, 4-aminobutyric acid, aspartic
acid, phenyl glycine, histidine, tryptophan, tyrosine, and phenylalanine,
wherein the phenyl group of phenylalanine or phenyl glycine may optionally
contain a single substitution selected from the group consisting of halo,
OR, and SR, wherein R is H or alkyl (1-4C) linked through a peptide bond
to the remainder of the compound;
wherein said compounds have diverse properties.
4. The panel of claim 3 wherein said diverse properties include diversity
in a property selected from the group consisting of hydrophobicity of X,
Hammett's Constants of X, and hydrophobicity of AA.sub.C.
5. A compound of the formula
##STR9##
or the alkyl (1-10C), alkenyl (1-10C), or arylalkyl (7-12C) esters or
amides or salts thereof;
wherein Z is selected from the group consisting of S, O and CH.sub.2 ;
wherein n is 1;
wherein X is a mono- di- or trisubstituted benzyl wherein said substitution
is selected from the group consisting of R', halo, NO, NO.sub.2, NH.sub.2,
OR, and SR, wherein R' is alkyl (1-6C), alkenyl (1-6C) or alkynyl (1-6C),
R is H or alkyl (1-6C);
Y--CO is .gamma.-Glu; and
AA.sub.C is phenylGly linked through a peptide bond to the remainder of the
compound.
6. The compound of claim 5 wherein Z is S and n is 1.
7. The compound of claim 6 which is a ester.
8. The ester of claim 7 which is an ethyl ester or a benzyl ester.
9. The ester of claim 8 which is a diester.
10. The ester of claim 7 which is selected from the group consisting of
.gamma.-Glu-Cys(p-ClBz)-phenylGly, .gamma.-Glu-Cys(octyl)-phenylGly,
.gamma.-Glu-Cys(Bz)-phenylGly, .gamma.-Glu-Cys(Hx)-r(-)-phenylGly,
.gamma.-Glu-Cys(Hx)-s(+)-phenylGly, .gamma.-Glu-Cys(decyl)-phenylGly,
.gamma.-Glu-Cys(p-NO.sub.2 Bz)-phenylGly, and
.alpha.-Glu-Cys(Hx)-phenylGly.
11. The ester of claim 10 selected from the group consisting of
.gamma.-Glu-Cys(p-ClBz)phenylGly, .gamma.-Glu-Cys(Bz)-phenylGly, and
.gamma.-Glu-Cys(p-NO.sub.2 Bz)-phenylGly.
12. The ester of claim 10 which is .gamma.-Glu-Cys(p-ClBz)-phenylGly.
13. The ester of claim 10 which is .gamma.-Glu-Cys(octyl)-phenylGly.
14. The ester of claim 10 which is .gamma.-Glu-Cys(Bz)-phenylGly.
15. The ester of claim 10 which is .gamma.-Glu-Cys(Hx)-phenylGly.
16. The ester of claim 10 which is .gamma.-Glu-Cys(decyl)-phenylGly.
17. The ester of claim 10 which is .gamma.-Glu-Cys(p-NO.sub.2
Bz)-phenylGly.
18. The ester of claim 12 which is the diethyl ester.
19. The ester of claim 13 which is the diethyl ester.
20. The ester of claim 14 which is the diethyl ester.
21. The ester of claim 15 which is the diethyl ester.
22. The ester of claim 16 which is the diethyl ester.
23. The ester of claim 17 which is the diethyl ester.
24. The compound .gamma.-Glu-Cys(Hx)(Me-I)-phenylGly and the alkyl (1-10C),
alkenyl (1-10C) or arylalkyl (7-12C) esters or amides.
25. The compound of claim 24 which is the ester.
26. The compound of claim 24 which is the diethyl ester.
Description
TECHNICAL FIELD
The invention relates to tripeptide compounds which are novel analogs of
glutathione. The invention also concerns panels of tripeptides that are
glutathione analogs which have diverse properties and are useful for
characterizing glutathione transferases (GSTs) and as solution phase
inhibitors of GST.
BACKGROUND ART
Glutathione (GSH), in its reduced form, is a tripeptide of the formula:
.gamma.-Glu-Cys-Gly. Reduced glutathione has a central role in maintaining
the redox condition in cells and is also an essential substrate for
glutathione S-transferase (GST) which facilitates the detoxification of
foreign substances by a number of mechanisms, including catalysis of the
coupling of an electrophilic portion of a toxin, for instance, to
glutathione, rendering the toxin more susceptible to clearance. A second
mechanism, which also involves glutathione as substrate, resides in the
reduction of peroxides with the concomitant oxidation of glutathione.
Adang, A. E. P., et al., Biochem J (1990) 269:47-54, described tripeptide
analogs of GSH which interact with various GST isoenzymes at different
concentrations. These analogs are modified forms of GSH in which at least
one of the glycine, cysteine, or gamma-glutamine residues is replaced by
an alternate amino acid residue.
Additional modified forms have been disclosed, for example, by Principato,
G. B., et al., Enzyme (1989) 41:175-180, who studied the effect of a
tripeptide GSH analog on glyoxalase II enzyme of rat liver. The tripeptide
used by this group was of the formula
.gamma.-Glu-.rho.-chlorophenylcarbonylmethyl-Cys-Ser. Morris, D., in
Biochem J (1960) 76:349-353, described the synthesis of
.gamma.-Glu-benzyl-Cys-Val. A large number of GSH tripeptide analogs
containing a substitution for only one of the three GSH amino acids have
been reported and are commercially available.
The invention described hereinbelow concerns novel glutathione tripeptide
analogs which are useful as affinity ligands on chromatographic supports
and as members of panels which are used to characterize the various
isoenzymes of glutathione-S-transferase. Glutathione-S-transferases (GSTs)
are present in the form of a number of isoenzymes which differ in specific
binding abilities, in substrate and inhibitor specificities and in tissue
distribution. Particular complements of GST isoenzymes, with their
accompanying differences in properties, thus are characteristic of
specific tissues or cell types, such as tumor tissues. As GST is central
to the overall metabolism of the tissue or cell as it relates to its
defense against toxic substances, the character of the complement of the
GST for the cell or tissue is important in designing strategies either for
the destruction of the cell or tissue, as would be desirable for tumor
cells, or for enhancement of its metabolic function, as would be the case
for normal tissue.
The various GST isoenzymes are dimeric proteins formed by binary
combinations of monomers encoded by at least fifteen known genes in four
gene families resulting in the theoretical possibility of several dozen
different dimers, even allowing for preferential dimerization of monomers
from the same gene family. In addition to the variability that arises from
these combinatorial possibilities, the GST isoenzyme subunits are
polymorphic in the human population and have been considered to be subject
to additional variation due to gene conversion events among the tandemly
repeated members of the family. Posttranslational modifications add
further to this variability. As each cell or tissue may contain one or
several of these theoretically possible enzymes, determination of the GST
complement is of great importance.
The present invention, by providing novel glutathione analogs which are
useful as sorbents and as solution phase inhibitors, as well as panels
that include them, offers improved methods to characterize and manipulate
individual GST enzymes or sets thereof.
DISCLOSURE OF THE INVENTION
The invention is directed to reagents useful in characterizing glutathione
S-transferase isoenzymes, in determining the GST complements of cells and
tissues, and in therapeutic applications. The invention compounds are
systematically modified forms of reduced glutathione and panels comprising
such analogs having diverse properties with respect to the targeted
enzymes. The invention compounds are also useful as chromatographic
affinity ligands, binding reagents, and enzyme inhibitors.
The esters of the tripeptides of the invention are particularly useful in
therapeutic and diagnostic contexts; the amides are also capable of
derivatization to moieties that can modify pharmacological properties or
physical behavior. Thus, in one aspect, the invention is directed to the
alkyl-type (1-10C), alkenyl-type (1-10C), and arylalkyl-type (7-12C)
esters or amides of a compound of the formula:
##STR3##
wherein Z is selected from the group consisting of S, O and C; n is 1 or
2; wherein when Z is S and n is 1, X is a mono- or disubstituted or
unsubstituted hydrocarbyl (1-20C) moiety optionally containing 1 or 2
nonadjacent heteroatoms (O, S or N), and wherein said substitution is
selected from the group consisting of halo, 'NO, --NO.sub.2, --NR.sub.2,
OR, and SR, wherein each R is independently H or lower alkyl (1-4C); and
wherein, when Z is S and n is 2, one X is as above defined and the other X
is lower alkyl (1-4C);
Y is selected from the group consisting of
##STR4##
wherein m is 1 or 2; and AA.sub.C is an amino acid coupled through a
peptide bond to the remainder of the compound of formula 1.
In another aspect, the invention is directed to a cycloamido form of the
compound of formula 1 as defined above; when Z is S, X can also be H.
In still another aspect, the invention is directed to a method to purify or
characterize a human glutathione S-transferase (GST) enzyme which
comprises contacting a sample thought to contain the enzyme with a solid
support to which is coupled a compound of formula 1, as defined above,
under conditions wherein the human GST is adsorbed to the solid support,
separating the support from the sample and eluting the human GST from the
support. In one preferred embodiment of this method, the support is
suitable for use in high-performance liquid chromatography (HPLC) systems.
In still other aspects, the invention is directed to methods to detect the
presence or absence of a GST enzyme in the sample, and optionally to
characterize it by class, which comprises treating the sample with a
compound of formula 1 as defined above and detecting the presence or
absence of a complex between any GST contained in the sample and the
compound of formula 1.
The invention also includes panels comprising at least five diverse
tripeptide glutathione analogs of formula 1 (or the esters, salts, amides
or cycloamido forms thereof) wherein the compounds in the panel have
diverse characteristics. These panels may be constituted as
chromatographic support panels and used to characterize a GST complement
of cells.
Finally, the invention is directed to compounds of formula 1 wherein X is a
mono-, di-, or trisubstituted mono- or bicyclic aryl (1-20C), preferably
benzyl, wherein the substitution is selected from the group consisting of
R', halo, --NO, --NO.sub.2, --NH.sub.2, OR, and SR, wherein R is H or
alkyl (1-6C), and R' is alkyl (1-6C), alkenyl (1-6C) or alkynyl (1-6C).
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1. Multivariate analysis of GST inhibitors. Inhibitor quality is
visualized by plotting potency against each of three recombinant human GST
isoenzymes (A1-1, M1a-1a, and P1-1) and projecting the points onto a plane
defined by the principal components of the distribution. Target values for
ideal inhibitors of each isozyme are denoted by circles labeled with the
isozyme name; the criteria for an ideal inhibitor are 100-fold specificity
and K.sub.i =0.1 .mu.M. Three families of compounds presented in Example 9
are compared: a selection of paralogs from Table 5 (numbers), a series of
n-alkyl GSH analogs from Table 7 (C.sub.n in boldface), and a selection of
the small organic molecules from Table 7 (italics). For purposes of
clarity, certain data are not plotted, namely, those for compounds of low
specificity, which are clustered at the center of the plot.
MODES OF CARRYING OUT THE INVENTION
The invention is directed to compounds that are useful individually as
chromatographic ligands and as GST inhibitors, as well as to panels of
related compounds which have diverse properties and which are useful in
determining profiles, for example, of GST enzymes, and in determining GST
complement in unknown samples. The invention compounds are systematically
modified forms of reduced glutathione and panels comprising such analogs
having diverse properties with respect to the targeted enzymes.
Previous tripeptide analogs of GSH, which interact with various GST
isoenzymes at different concentrations, are modified forms of GSH in which
at least one of the glycine, cysteine, or .gamma.-glutamine residues is
replaced by an alternate amino acid residue. See, for instance, Adang, A.
E. P. , et al., Biochem J (1990) 269:47-54. Given the variety of
nonstandard amino acids that have been substituted for each amino acid in
the GSH tripepcide and the numerous other possibilities, combined with the
variety of substrates that could be linked to the cysteine sulfhydryl, it
would be possible to contemplate tens of thousands of GSH analogs as GST
inhibitors.
Following the previously described paralog strategy (U.S. Pat. Nos.
4,963,263 and 5,133,866, the disclosures of which are incorporated herein
by reference), a systematic sampling of this potential set has been
prepared. Thus, a representative sampling of the potential diversity is
achieved through monomer choices resulting in concurrent variations across
a wide range for multiple parameters relevant to binding to GSTs from
several. mamalian species, primarily the rat. Parameters which have been
emphasized include hydrophobicity, size, and electronegativity. Variations
in properties were created primarily at the C-terminus and sulfhydryl
positions.
When tested by affinity chromatography several of these diverse sorbents
selectively bind mammalian GST isoenzymes of a particular class or classes
according to the following classification scheme proposed by Mannervik,
B., et al., Proc Natl Acad Sci (1985) 82:7202-7206: Alpha (e.g. human a
and A1-1, rat 1-1 and 2-2), Mu (human .mu., M1a-1a, rat 3-3 and 4-4), Pi
(human .pi. and P1-1, rat 7-7) and, a more recently proposed class, Theta
(rat 5-5). When tested for inhibition of GST enzymatic activity, several
sorbents also act as selective or specific inhibitors of GST isoenzyme
classes. Therefore, the compounds, panels and methods of this invention
enable novel strategies for the design of drugs targeting cells with
elevated levels of particular GST isoenzymes.
The Compouncs of the Invention
The novel compounds of the invention include the moiety of formula 1,
wherein one X is a mono- or disubstituted or unsubstituted hydrocarbyl
(1-20C) moiety optionally containing one or two nonadjacent heteroatoms
(O, S or N), and the other is not present or is lower alkyl (1-4C). In
preferred embodiments n is 1 and X is unsubstituted hydrocarbyl. Preferred
embodiments for X include methyl, propyl, hexyl, octyl, benzyl, naphthyl
and trityl. In the cycloamido forms, X may also be H.
As used herein, "hydrocarbyl" refers to a straight or branched chain or
cyclic, saturated or unsaturated, aliphatic or aromatic hydrocarbyl
residue containing 1-20C. In addition to the 1-20C, where structurally
realistic, the hydrocarbyl may also contain one or two nonadjacent
heteroatoms which are O, S or N. Thus, the hydrocarbyl group so modified
may be an ether, a diether, a thioether or a dithioether, or a secondary
or tertiary amine or diamine. Representative of such substituents include
methyl, ethyl, propyl, isopropyl, butyl, tertiary butyl, hexyl, octyl,
nonyl, 2,3-dimethyloctyl, dodecyl, 9,9-dimethylundecyl, allyl, 2-butenyl,
isobutenyl, cyclohexyl, cyclopentyl, cycloheptyl, phenyl, benzyl,
4-methylbenzyl, triphenylmethyl, methoxyethyl, ethylthioethyl,
diethylaminopropyl and the like. Also included in hydrocarbyl groups
suitable for X are bicyclic aryl (1-20C) moieties including naphthyl and
heterocyclic relatives and the like.
In addition, the hydrocarbyl or hydrocarbyl containing one or two
heteroatoms may optionally be substituted by one or two substituents.
These substituents may be halo, i.e., fluoro, chloro, bromo or iodo, or
hydroxy or sulfhydryl, and/or alkyloxy or alkylthio, such as methylthio,
butylthio, propoxy or ethoxy, and/or --NO, --NO.sub.2, or --NH.sub.2,
--NH(alkyl) or --N(alkyl)(alkyl).
AA.sub.C may be any gene-encoded amino acid. AA.sub.C may also be an amino
acid residue which is not encoded by the gene, such as hydroxyproline
(HP), 4-aminobutyric acid (4ABu), .beta.-alanine (.beta.-ala or .beta.A),
phenylglycine (PG or .phi.G), and the like. AA.sub.C is bound to the
remainder of the compound of formula 1 through a peptide bond.
In the compounds of the invention, substitution of phenylglycine for Gly,
increasing the hydrophobicity and bulk of this site, results in high
specificity for .pi.-class isoenzymes (e.g., rat 7-7). The use of Ala and
.beta.-Ala at the Gly site produces sorbents with high specificity for
.mu.-class isoenzymes (e.g., rat 3-3 and 4-4). The shift from Ala to
.beta.-Ala, an alteration that increases the length of the backbone,
results in differential binding of different .mu.-class isoenzymes. The
Gly substituents do not determine the total character of the sorbent,
however. For example, the use of a hexyl adduct at the Cys site, combined
with Ala or .beta.-Ala at the Gly moiety, greatly diminishes GST binding.
For compounds of the invention whsubstituted substituted benzyl, whether in
the form of the free acid or salt of formula 1 or as esters, amides or
cycloamido forms, specificity for various subtypes of human GSTs can be
controlled by varying the para substituent on the benzyl moiety.
Embodiments of X which contain hydrophobic para substituents such as
t-butyl or methyl or benzyl, bind preferentially to human GSTs of the .mu.
(M1) class, such as GST-M1a-1a. Electron withdrawing substituents at the
para position, such as nitro or chloro preferably bind .pi. (P1-1)
enzymes. Plots of Hammett sigma/meta values against the log fraction of
isoenzyme bound show a linear correlation but no clear correlation appears
when sigma/para values are used. Some steric bulk appears necessary to
bind GSTs generally; benzyl substituents which are unsubstituted or
contain only fluoride substitutions do not bind appreciably.
For compounds of the invention wherein X is an unsubstituted hydrocarbyl
(1-20C) moiety, whether in the form of the free acid or salt of formula 1
or as esters, amides or cycloamido forms, specificity for various subtypes
of human GSTs can be controlled by varying the hydrophobicity of the
substituent on the sulfur atom of the Cys moiety, for instance, the length
of n-alkyl adducts. Generally, the binding strength for GSTs increases
with the chain length of the alkyl group, but there is a change in
isoenzyme selectivity with increasing chain length. Embodiments of X which
contain S-alkyl chains of three or four atoms inhibit preferentially human
GSTs of the .mu. class, particularly GST-M1a-1a. In the range of alkyl
groups containing five to eight carbons, human GSTs of the .alpha. and
.mu. classes are inhibited to about the same extent and are selectively
inhibited as compared to human .pi.-class isoenzyme. Finally, for S-nonyl
and S-decyl GSH derivatives, human GSTs from all three classes are
inhibited to about the same extent.
The compounds of the invention may include the alkyl, alkenyl or arylalkyl
esters or alkyl-type, alkenyl-type or arylalkyl-type amides, or may be in
the amidocyclic forms. Alkyl-type esters of the free carboxyls are esters
of the straight- and branched-chain and cyclic alkyl alcohols (1-10C) such
as methanol, ethanol, isopropanol, t-butanol, n-hexanol and the like. The
carbon chain of the alkyl-type alcohol may be interrupted by one or two
nonadjacent heteroatoms, i.e., N, O or S. For example, alkyl-type alcohols
include N,N-dimethylethanolamine and
2-(1-hydroxy-2-ethyl)-4,4,6-trimethyltetrahydro-1,3-oxazine. Suitable
alkyl-type (1-10C) amides are those of primary straight- or branched-chain
or cyclic alkyl amines, such as methylamine, ethylamine, n-propylamine,
isopentylamine, and isohexylamine. These carbon chains also may be
interrupted by one or two nonadjacent heteroatoms, i.e., N, O or S.
Alkenyl-type esters and amides are prepared from the corresponding amines
which contain at least one double bond. Arylalkyl-type esters may contain
7-12C and include, phenyl-lower alkyl derivatives such as benzyl,
2-phenylethyl, 2-phenylpropyl, and the like. The phenyl group may be
unsubstituted or may be substituted with 1-2 substituents, such as methyl,
chloro and the like which do not materially effect the properties of the
invention compounds. Again, the hydrocarbon chain may be interrupted with
1 or 2 heteroatoms, and heterocyclic aromatic systems also may be
included. The esters and amides are prepared using conventional
techniques, with suitable protection of any alcohol or amino functional
groups in the substrate compound of formula 1.
It should be noted that the compounds may be prepared as either the mono-
or diesters or the mono- or diamides (or triesters or triamides, depending
on the choice of AA.sub.C). Thus, the invention includes esters wherein
only one of the carboxyl groups is esterified, where two carboxyl groups
are esterified or where all carboxyl groups (if more than two) are
esterified. Similar embodiments are applicable to the corresponding
amides. In addition, the compounds may be prepared as mixed esters/amides
wherein one carboxyl is an ester and another an amide.
Salts of the compounds of the invention may be formed of inorganic bases
such as sodium or potassium hydroxide or of organic bases such as caffeine
or piperidine to form the basic salts of the free carboxyl groups or may
be formed from organic or inorganic acids to obtain the acid addition
salts of free amino groups. Thus, the salts may be of inorganic bases such
as sodium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium
hydroxide, and the like, or of organic bases such as trimethylamine,
pyridine, pyrimidine, lysine, caffeine, and the like. The acid addition
salts may be formed from inorganic acids such as hydrochloric acid,
hydrobromic acid, sulfuric acid, phosphoric acid, and the like, or from
organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic
acid, oxalic acid, malic acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, salicylic acid, and the like.
Salts are formed in standard protocols by treating with the appropriate
base or acid at a temperature of from about 0.degree. C. to about
100.degree. C., preferably at room temperature either in water alone or in
combination with an inert water-miscible organic solvent such as methanol,
ethanol or dioxane.
Cyclic forms of the compound of formula 1 are prepared by bridging free
amino and free carboxyl groups contained in the substrate compound.
Formation of the cyclic compounds is conducted conventionally by treatment
with a dehydrating agent such dicyclohexyl carbodiimide by means known in
the art per se, with suitable protection if needed.
In most embodiments of the compounds of formula 1, the cycloamide is formed
between the free amino group present at the amino terminus on Y with the
carboxyl group at the carboxy terminus represented by AA.sub.C. However,
in those limited embodiments wherein AA.sub.C contains a side-chain amino
group (such as those wherein AA.sub.C is lysine) alternative forms of the
cycloamido compounds may also be prepared involving the side-chain amine
of AA.sub.C and the side-chain carboxyl of Y. Suitable protecting groups
can be used to direct the cycloamidation as is known in the art.
The compounds of the invention can be further characterized by designating
the identities of Y, n, X, Z and AA.sub.C. In order to designate Y, the
carbonyl group shown adjacent the Y substituent in formula 1 is
conveniently included in the designation; if this is the case, the various
embodiments of "Y--CO" include .gamma.-Glu, .beta.-Asp, Glu, Asp,
.gamma.-Glu-Gly, .beta.-Asp-Gly, Glu-Gly, and Asp-Gly, which in the
one-letter amino acid codes can be symbolized as .gamma.E, .beta.D, E, D,
.gamma.E-G, .beta.D-G, E-G, and D-G, respectively.
The various substituents designated by X can be noted by standard
abbreviations, and their inclusion in the tripeptide analogs of the
compounds of the invention symbolized by C(X) when n=1 or C(X)(X) when
n=2. In those compounds of formula 1 wherein n equals 2, the sulfur of the
cysteine residue will have a positive charge and exist as a sulfonium ion.
Z is selected from the group consisting of S, O and C. Compounds in which Z
is O include, for example, .gamma.-glutamyl-(o-hexyl)serinyl-glycine,
.gamma.-glutamyl-(o-octyl)serinyl-glycine and the like. Compounds in which
Z is C include, for example, .gamma.-glutamyl-histidinyl-glycine,
.gamma.-glutamyl-(.pi.-benzyl)histidinylglycine, and
.gamma.-glutamyl-.alpha.-aminooctanoyl-glycine, and the like. Compounds in
which Z is O or C are more resistant to oxidation than compounds
containing S at the Z position.
The notation for AA.sub.C may also be in the standard one-letter amino acid
abbreviation code or other suitable abbreviation for non-gene encoded
amino acids.
Thus, using this notation, suitable compounds of formula 1 include:
.gamma.E-C(Bz) (Me)-G,
.beta.D-C(Trt)-A, .gamma.E-C(p-ClBz)-G,
E-C(Pr)(Me)-V, .gamma.E-C(p-NO.sub.2 Bz)-PG,
.gamma.D-C(Hx)-PG, .gamma.E-C(p-MeBz)-V,
E-G-C(Bz)(Et)-4ABu, .gamma.E-C(p-BuBz)-.beta.A,
D-G-C(Hx)-F, .gamma.E-C(p-MeOBz)-.beta.A,
.gamma.E-C(Hx)(Me)-N, .gamma.E-C(p-EtSBz)-PG,
.gamma.E-C(Trt)-N, .gamma.E-C(p-Me.sub.2 N-Bz)-.beta.A,
.gamma.E-C(o-BuBz)-PG
and the like.
These compounds of formula 1 are cyclized or esterified/amidated to obtain
the compounds of the invention.
Features of the Invention Compounds
The compounds of the invention are useful as solution-phase or immobilized
inhibitors of enzymes, such as glutathione-S-transferases (GSTs), which
utilize glutathione as substrates. Such inhibition may be desirable both
in analytical and therapeutic contexts.
As therapeutic agents, the esterified compounds of the invention are most
useful since permeation of the cell membrane is best achieved by these
less hydrophilic forms. Particularly preferred esters are the benzyl
esters and derivatized benzyl esters. Also preferred are the ethyl esters.
Thus, for use in vitro in cell culture, for example in screening assays,
the preferred compounds of the invention are the esterified forms. In
addition, both the esters and amides offer the opportunity to modify the
invention compounds in ways that affect their suitability as therapeutic
agents, as diagnostic reagents, and as ligands for coupling to solid
supports. The ester or amide groups may contain additional useful ligands
such as hydrophobic moieties to enhance cell permeation, reactive groups
to facilitate linkage to other substances, and additional ligands such as
antibodies or fragments thereof, carriers to enhance immunogenicity and
the like. Further, the compounds can be modified by including substituents
such as chelating agents which facilitate purification by immobilized
metal ion affinity chromatography or substituents which alter the
solubility of the peptide or which affect pharmacokinetics such as the
addition of polyethylene glycol.
Thus, the esters and amides of the compound of formula 1 offer distinct
advantages over the free acid or salt forms in a variety of contexts.
The cycloamido forms of the compound of formula 1 offer the advantage of
enhanced specificity and selectivity among target GSTS. The cyclic forms
in general are superior as chromatographic ligands to the open chain
forms. In addition, their inhibition characteristics are modulated by this
feature.
The compounds of the invention that contain substituted benzyl as the
embodiment of X are advantageous in that they offer the opportunity
systematically to control the selectivity of the compounds for the various
GSTs. For instance, as shown in Example 8 herein, the binding specificity
among the .mu. and .pi. groups of human GSTs is controlled by the nature
of the para-substituent on a benzyl moiety attached to the sulfur atom of
the Cys residue of certain compounds of the invention. There is a
correlation in binding specificity with sigma (meta) values of the
para-substituents on S-benzyl Cys moieties.
Use as Chromatographic Ligands and Analytical Reaaents
The invention compounds also are useful individually as diagnostic
chromatographic supports and chromatographic tools. The use of these
compounds in affinity chromatography, for characterization and for
purification, as well as in diagnostic assays, is particularly important
in the case of human GSTs. While panels of the invention compounds are
most convenient in determining the GST complement of tissues, individual
compounds can be used to detect the presence of GSTs in general, or of
GSTs of a particular type in humans, as well as in purification of
particular types of human GSTs. Thus, as shown in Example 8 below, various
compounds of the invention have different propensities for binding human
GSTs of the .mu. (M1) and .pi. (P1) classes, as well as various rat
isoenzymes. In addition, as shown in Example 9, compounds of the invention
also show selective solution phase inhibition of particular human GSTs. In
most cases affinity chromatography and inhibition studies show the same
enzyme specificity for each compound of the invention.
The compounds of the invention, or the compounds of formula 1 alone as the
free acids or salts, can be used as chromatographic ligands for
purification and characterization of human GSTs collectively, for
purification and identification of human classes of GSTs or for
purification or separation of individual human GST enzymes, depending on
the choice of ligand.
For use as affinity ligands on chromatographic support, the compounds that
include formula 1 are coupled to a suitable solid matrix, such as
Sepharose, polyacrylamide, silica, and the like using standard coupling
techniques. The noncyclic forms of the compounds of the invention contain
at least amino and carboxyl functional groups which can be derivatized to
suitable linkers or directly to supports. Depending on the nature of the
solid support, the coupling of the affinity ligand may employ direct
covalent coupling or may require the use of homo- or heterobifunctional
linkers such as those available from Pierce Chemical Company (Rockford,
Ill.). It may also be desirable to distance the affinity ligand from the
surface of the support in order to render it more accessible. Such
distancing can be accomplished using spacer arms, as is generally
understood. Further, the support may be treated with an inert material,
such as, for example, human serum albumin, so as to minimize unwanted
interactions, especially if the support is to be used for the
chromatography of biological samples.
The derivatized chromatographic supports may take advantage of the
diversity of the compounds which include the structure of formula 1 by
coupling the solid support to more than one such ligand. The ligands can
be arranged in a gradient, at random as a mixture, or in any predetermined
pattern desired. In addition, combinations of the compounds of the
invention can be used in chromatographic applications wherein a balance is
achieved between one or more compounds which serve as affinity ligands and
one or more compounds which are used as competitors to the affinity ligand
to effect elution of the materials subjected to chromatographic separation
or characterization. Materials of varying affinity for the moieties
subjected to the chromatographic technique are used for the ligands and
the eluting agents, as illustrated in Example 10, below.
The chromatographic supports derivatized to compounds that include formula
1 may then be used for preparative separation of human GSTs or other
enzymes for which the ligands have affinity, other enzymes utilizing
glutathione as substrate, antibodies which react with glutathione, or
other moieties which bind with moderate affinity to the ligands.
Chromatographic supports coupled to the compounds of the invention may
also be used in diagnosis to determine the presence, absence, quantity or
nature of materials in biological or other samples suspected to contain
materials having similarity in structure to glutathione. For instance,
affinity chromatography using compounds of the invention in a novel HPLC
method can be used to isolate and separate GST isozymes in dimeric,
bioactive form from human tissues such as liver, as illustrated in Example
10, below.
The suitable compound containing the structure of formula 1 useful in such
separations or analyses may be evident from the nature of the analyte or
material whose preparation is desired, or may readily be determined by
preparing a diverse panel of the compounds and screening the panel for the
most effective affinity ligand.
The compounds containing the structure of formula 1 may also be used for
detecting the presence or absence of human GSTs in general or for a
particular class or isoenzyme thereof. Protocols for such assays mimic
those used in immunoassay; these protocols are generally applicable to any
assays which involve specific binding partners where the specificity of
the assay depends on the specificity of the binding. Thus, in such assays,
the compounds including the structure of formula 1 play the role of
antibodies or other immunoreactive agents specific for the analyte, in
this case the relevant GST. Such protocols include sandwich assays,
competition assays, direct binding assays and the like with a wide variety
of detection means for formation of "immunocomplexes" (in this case a
complex between GST and the compound containing the structure of formula
1). Such detection means include florescent tags, enzyme labels,
radioactive labels and combinations of these.
Panels
The panels of the invention are constructed of at least five diverse
tripeptide analogs of glutathione of formula 1, wherein X, AA.sub.C and Y
are as defined above but wherein X can also be H. The panels are most
useful when maximally diverse in character. This diversity can be supplied
by varying the natures of Y, AA.sub.C and X. In general, by using X
substituents, for example, of varying hydrophobicity, a range of such
hydrophobicity characteristics can be obtained. X is also a convenient
substituent for variation of the Hammett Constants (sigma/meta and
sigma/para) diversity of the resulting compounds. The diversity resulting
from variation of AA.sub.C is somewhat less focused; that provided by
variations in Y is limited by the limitations on that structure.
The Hammett Constants refer to the values analogous to those obtained
showing the electronic influence of substituents on the ionization
constant of benzoic acid. As a result of this early work, numerical values
have been assigned to a large array of different substituents. Tables of
such values for various substituents are given, for example, by Ritchie,
C. D. et al., Prog Phys Org Chem (1964) 2:323.
The construction of a series of the analogs of the invention can be made
which systematically varies the value of this parameter by, for example,
utilizing as the embodiment of X, a benzyl group which is further
substituted with an additional substituent at the para position, such as
nitro, chloro, methoxy, or methyl.
The steric characteristics and the resulting properties of the compounds of
formula 1 which are members of the panel can also be controlled by
cyclization of one or more members of the panel.
For example, a two-dimensional matrix of the glutathione analog tripeptides
of the invention forming a suitable two-dimensional panel can be
constructed by varying the AA.sub.C component from a small hydrophobic
residue to a large hydrophobic residue to a positively charged residue to
a neutral residue and finally to a negatively charged residue, thus
influencing, in the latter three analogs, the inductive electronic effect.
Similarly, the X substituents can be varied in the second dimension over
the same range from small hydrophobic to large hydrophobic to positively
charged to neutral to negative. The resulting matrix provides a suitable
panel for use in, for example, determining suitable compounds to use as
adsorbents.
Particularly useful embodiments of X are the substituted benzyls wherein it
can be shown that variation of the hydrophobicity and electron donating or
withdrawing capacities of the para substituent has a systematic
correlation with ability to bind certain classes of human GSTs.
Use of the Invention Panels
The panels are useful in determining the differing complements of the
glutathione-S-transferase (GST) isoenzymes as they occur in normal (as
compared to unwanted) cells or tissues. By "GST complement" is meant the
pattern of levels of GST isoenzymes that is present in such cells or
tissues or which is genetically programmed in such a manner that induction
or repression of expression levels of such GSTs is manipulable. As
explained in the Background section above, GSTs are homo- or heterodimers
formed from subunits, of which at least seven are well known. In addition,
although these isoenzymes have been classified broadly, individual members
within each class may differ from individual to individual due to genetic
variation. The properties of the various isoenzymes differ with respect to
a series of measurable parameters including substrate specificity,
susceptibility to inhibition, binding to specific reagents, and
inducibility of expression.
Use of the Invention Panels to Determine GST Profiles--Background
Perhaps the most easily understood approach to determining the GST
isoenzyme complement of an unknown tissue sample presumes a reference set
of all GST isoenzymes which have reactivity profiles that have been or can
be determined. Thus, assuming a high enough resolution separation, for
example, using any separation technique, analogous, for example, to high
resolution chromatographic focusing, an elution pattern is referenced to a
series of known enzymes. Other methods for separating and characterizing
the known isoenzymes could include the use of antibodies that have been
prepared to specific isoenzymes, such as those established for several GST
human isotypes and used to assess GST content of these isotypes in
candidate tissues (Howie, A. F., et al., Carcinocenesis (1990) 11:451-458;
Beckett, G. J. Clinica Chimica Acta (1984) 141:267-273). Gel
electrophoresis separations can also be used. The location of the GST
bands following electrophoresis under nondenaturing conditions can be
determined, for examples, by the method of Ricci, G., et al., Anal Biochem
(1984) 143:226-230. The location of various isoenzymes resulting from
chromatographic separations can be detected using substrates common to all
isoenzymes, such as 1-chloro-2,4-dinitrobenzene (CDNB). Indeed, the
distribution of activity as assayed by CDNB in various tissues has been
conducted by Pickett, C. B., and Lu, A. Y. H., Ann Rev Biochem (1989)
58:743-764.
The use of these direct separation methods to obtain a pattern of GST
isoenzyme distributions in cells and tissues of interest can be used to
obtain a GST complement for such cells and tissues that may be useful in
the design of therapy, provided that each of these isoenzymes has a
reactivity profile which has been determined previously following
separation techniques permitting isolation of the individual isoenzyme
with retention of activity. Such a reactivity profile would take account
of the substances which are effective substrates, substances which are
effective inhibitors, and substrates which are effective inducers or
activators of GST activity. Once the GST isoenzyme is identified and
quantitated by virtue of its position in the elution pattern or
electrophoretic gel, for example, reference is made to the reactivity
profile of the known and previously isolated isoenzyme in order to predict
or design treatment protocols.
This method, while readily comprehensible, is not practical due to the
large number of GST isoenzymes that are potential candidates for inclusion
in the complement and due to the mutability of the repertoire of GST
isoenzymes per se. Thus, a polymorphism in the population of available GST
isoenzymes is likely to result in a protein with unaltered mobility, for
example, in the elution pattern, but with altered substrate specificity or
inhibition pattern, or vice versa. In either case, the results of the
matching of the position in the elution pattern to the set of reference
characteristics would give misleading results.
A somewhat improved result can be obtained by utilizing multiple separation
techniques, it being less likely that mobility would be unaffected in
multiple separation systems as compared to one. Such a system is
generically illustrated by a simple hypothetical system in which a first
sorbent, P1, produces four isoenzyme peaks in its elution pattern and five
are obtained on a second sorbent, P2. If the substrate specificity
patterns (for substrates A, B and C, for instance) indicate that a given
peak from P1 (identified, e.g., as 1.2) is substantially the same in
substrate profile as a particular peak separated on sorbent P2 (peak 2.4),
then one would assume that a tissue sample which provided a peak at 1.2 in
sorbent P1 and at 2.4 in sorbent P2 would be highly likely to have a
reactivity profile (with A, B and C) the same as that of the isoenzyme
forming peaks 1.2 and 2.4.
The technique illustrated in this hypothetical system can be applied using,
as affinity-based sorbents, the novel glutathione tripeptide analogs of
the invention or one such novel tripeptide in combination with an
additional analog or panels of analogous glutathione analogs with diverse
properties. It is believed that to perform effective characterization, a
panel of at least two, and preferably three, and more preferably 5 such
analogs should be used. The members of the panel should have properties
which are sufficiently diverse to assure discrimination among the various
GST isoenzymes in the complement.
If the separation technique preserves enzymatic activity, the reactivities
of each enzyme against potential drugs can be directly determined.
Nondenaturing separations in the art, however, suffer from either a lack
of resolution or from hypersensitivity to structural changes, making peak
identification too problematic for effective guidance of therapy. Ion
exchange chromatography, for example, can be used as a step to purify
individual GST isoenzymes, but has inadequate or inappropriate resolution
as an analytical tool. IEF, another technique available in the art, is
prone to generation of numerous extraneous peaks due to in vivo or in
vitro posttranslational modification of the protein, and there is no
necessary linkage between such structural changes and functional
variation.
Thus, determination of the activity profile of the GST complement in cells
or tissues by separating the individual isoenzymes using prior art methods
and determining an activity profile for each of them against all possible
chemotherapeutic drugs would be laborious, but is enhanced by the
availability of the novel GSH-analog tripeptides of the invention. The
compounds of the invention permit separation without denaturation of the
GST enzymes.
A more efficient approach takes advantage of profiles of GST isoenzyme
complements which simultaneously measure specific binding activity and
reactivity characteristics. These profiles, designated survey of
characteristics profiles, or "SC" profiles, permit the determination of a
reference set of SC profiles which include information on substrate
specificity, induction in response to specific inducers, and the like, as
well as additional binding or electrophoretic mobility characteristics. By
applying computational methods to comparison of these profiles, the
requisite information for the design of therapeutic modulators and
accompanying protocols and for prediction of success or failure of
proposed protocols can be obtained for significantly larger numbers of
specimens than by prior art methods, as is needed to provide an adequate
guide for therapy. Among the parameters that can be used for obtention of
an SC profile is the ability to bind to members of the panels of
tripeptide GSH analogs of the invention, or the effect of such panel
members on activity.
In this approach to determining the GST complement of unknown samples of
cells and tissues, advantage is taken of pattern recognition techniques.
In connection with this approach, what is here termed an SC profile is
determined generally with respect to a panel of reagents which react
specifically and differentially with the various GST isoenzymes. This is
analogous to the determination of profiles obtained by cross-reaction
immunoassay, and refers to any pattern of reactivity of a candidate GST
isoenzyme or mixture of isoenzymes with a panel of reagents. Thus, the SC
profile may be determined with respect to turnover rates for various
substrates; effective levels of inhibitor concentration for various
inhibitors; levels of inducers required to induce the expression of the
gene for the GST isoenzyme in the context of a particular host cell;
mobility in electrophoresis in the presence or absence of inhibitors or
substrates; elution times from paralog or other affinity columns or,
indeed, the classical pattern of binding with a panel of antibodies. The
SC profiles obtained for individual GST isoenzymes or mixtures of
isoenzymes at various concentration levels can be manipulated in various
ways, described herein, to provide a reference set against which SC
profiles of unknown samples can be compared.
In general, the SC profile will provide values for each of a panel of
"information channels" wherein each information channel describes a
characteristic of the GST complement or standard, such as the binding
affinity for an antibody, a substrate affinity, an elution time or the
like. At least some of the information channels should relate to values
that vary with concentration of the GST.
Determination of the GST complement for cells or tissues is useful per se
in diagnosis and characterization of samples. In addition, for use in the
design of embodiments of the strategies to impair or destroy unwanted
tissue, the SC profiles must provide information related to GST activity
so that activity differences between normal and unwanted tissues can be
determined. Thus, the SC profiles of the unwanted tissue must be readily
comparable to the reference standards which, in turn, must at least in
part be based on reactivity patterns that will aid in the design of
therapeutic modulators and the selection of drugs or prodrugs. For this
application, at least a portion of the reference profiles must be grounded
in substrate turnover rate data, inhibition data, or data relating to
induction of isoenzyme production level, or any other reactivity which
will permit manipulation of the GST isoenzyme in situ. The panels of the
invention may be used to determine such effects on activity. The
combination of chromatographic separation which permits activity to be
retained using the novel compounds of the invention along with preparation
of SC profiles with regard to reactivity-affecting reagents for these
standards is one approach to obtaining the needed data.
The SC profiles of the reference standards and of the unknown samples are
determined with respect to panels of "specifically reactive reagents."
These reagents may include a variety of substances, including paralogs,
substrates, inhibitors, inducers, antibodies, as well as "reagents" which
are actually techniques such as gel electrophoresis or affinity
chromatography where the extent of reactivity is determined as
electrophoretic mobility or elution time. Thus, "specifically reactive
reagents" is not limited to those reagents which effect a chemical
reaction, but includes any reagent or technique that permits a
characteristic parameter to be obtained for the sample with respect to
that reagent.
Of course, the panels of the analogs of the invention can be used as
"specifically reactive reagents" either as binding agents, chromatographic
supports, or as inhibitors of enzyme action in solution. The comparative
ability of these compounds as members of the panel to inhibit the
enzymatic reactions catalyzed by GST can be used as a characteristic SC
profile, as can elution patterns from columns containing the tripeptide
analogs as affinity ligands.
Determination of Reference SC Profiles
While reference standards for some purposes, as described below, can be
prepared directly from normal tissue of a particular subject, it is also
useful to provide a databank of SC profiles for a variety of previously
isolated GST isoenzymes with characteristic reactivity patterns. By
matching these reactivity patterns with those from biopsy samples of the
unwanted tissue, the appropriate design for therapeutic modulators and
choice of prodrugs or toxins can be made.
U.S. Pat. Nos. 4,963,263 and 5,133,866, the disclosures of which are
incorporated herein by reference, describe panels of paralog affinity
reagents that are useful in chromatographic separations of closeparations
of closely related substances. The panels of the invention are similarly
diverse. Paralog-type panels using the GSH tripeptide analogs of the
invention can be conveniently used in the preparation of affinity supports
for the separation of various GST isoenzymes in purified form while, in
each case, retaining the activity of the native isoenzyme. Unlike the
reverse-phase HPLC or Western blot methods of the prior art, the separated
isoenzymes prepared using chromatography based on affinity for the
compounds of the invention behave in a manner virtually precisely similar
to that of the isoenzymes as they occur in nature. For each such purified
isoenzyme, then, a SC profile with respect to reactivity of substrate or
other activity-affecting reagent such as those represented by the
compounds of the invention can be constructed. A helpful data bank of a
large number of SC profiles characteristic of these purified isoenzymes
can then be retained and stored in mathematically or computationally
accessible form for comparison to samples obtained from the unwanted
tissue.
Panels of tripeptide glutathione analogs, at least one member of which
contains the structure of formula 1, can be used as the basis for the
collection of information channels which provides the SC profiles for the
reference set. Conjugation of known isoenzyme specific substrates to the
members of the panel or conjugation of the members of the panel directly
to sorbent further increases the systematic diversification of binding
properties. Profiles for standards and unknown samples can be obtained and
compared using the panels of the invention in appropriate configurations,
such as affinity columns. Thus, particularly useful are panels of diverse
tripeptide analogs including at least one analog containing formula 1
wherein X is a mono- or disubstituted or unsubstituted hydrocarbyl (1-20C)
moiety optionally containing 1 or 2 nonadjacent heteroatoms (O, S or N),
and wherein said substitution is selected from the group consisting of
halo, NO, NO.sub.2, NR.sub.2, OR, and SR, wherein R is H or lower alkyl
(1-4C) and
AA.sub.C is an amino acid coupled through a peptide bond to the remainder
of the compound of formula 1.
Determination of GST Complement
In general, two different approaches can be made to determine the GST
complement of an "unknown" sample cell or tissue. First, as described
above, using general techniques presently practiced in the art, the
individual isoenzymes contained in the sample can be separated using
affinity supports and tested individually for their patterns of
activities. The individual isoenzymes from the sample can be obtained and
then independently assessed for their substrate specificity, inhibitor
specificity and for identifying substances which induce the activity or
production of the isoenzyme.
In a second, less laborious, approach, pattern recognition techniques are
employed to obtain an instant readout for samples of either or both
unwanted and normal tissue for an individual subject by matching these
patterns against a reference set prepared as above. In this approach, less
volume of sample is required and no separation is necessary. This method
is also useful when applied to tissue slices using histochemical staining
for GST activity.
With respect to the first approach, a modification of the separation method
used by Vos, R. M. E., et al., Biochem Pharmacol (1988) 37:1077-082, can
be used. In this method, the cytosol fraction from a complete rat liver
was subjected to an affinity column of S-hexyl GSH Sepharose used as an
affinity reagent for GSTs as a group. The eluted GST mixture was
concentrated and separated by chromatofocusing on a mono-PHR 5/20 column
(Pharmacia FPLC system). The individual isoenzymes were collected in
separate fractions and analyzed. Fractions were identified by their
position in the elution profile, their subunit molecular weight, and
specific activities toward 1-chloro-2,4-dinitrobenzene, which is a
substrate for most known GSTs.
By using as the affinity ligand a paralog chosen from among the compounds
of the invention, milder conditions can be employed, and more active forms
of the GST isoenzymes can be recovered. These are then tested for
substrate specificity, etc.
As described above, one might consider the possibility of simply using
arbitrary separation technology such as that of Vos that provides an
elution pattern characteristic of the various GST isoenzymes, and matching
the elution pattern for cells or tissue of unknown GST complement with the
preset elution pattern to determine the nature of the GST complement in
the unknown. One problem with this approach, however, lies in the genetic
mutability of GST, so that it is difficult to make reliable matches that
will retain the inferred characteristics and thus be assured to have
similar reactivity patterns. The genetic mutability of the isoenzymes as
well as their sensitivity to posttranslational modifications is very
likely, in any particular case, to have a profound effect on the substrate
specificity, inhibition patterns, and the like, as well as in binding and
physical characteristics, such as pI. There is no guarantee that a
correlation will exist between reactivity variation and physical property
or binding variation; indeed, the probability is that the effects will not
be correlated. As described above, this problem can be mitigated by using
multiple affinity reagents, also provided by the invention compounds.
In the pattern-matching approach a more reliable assay is conducted by
comparing the profile of reactivity of an unknown sample with a set of
reference SC profiles. The application of this approach to the
determination of the composition of analyte in general is described in
copending application Ser. No. 07/678,049, filed 2 Apr. 1991, the
disclosure of which is incorporated herein by reference. According to the
techniques described in this application, a predetermined plot of profiles
obtained from samples of known analyte composition is used as a reference
with which an SC profile of the sample to be tested can be compared.
Generally, a panel of 2-10, preferably 4-6, different specifically
reactive reagents is first used to provide profiles for samples of known
compositions. In the referenced application, specific binding assays were
used where there was cross-reactivity by the candidate analytes across a
panel of binding agents, and the profiles were obtained by measurement of
inhibition values for binding of a known binding partner by various
analytes. The collection of profiles is then treated mathematically by any
of a number of techniques to generate a readable comparison with the
corresponding SC profile of an unknown sample.
For use in determining the GST complements needed to practice the
therapeutic methods of the invention, the analogous SC profiles can be
determined using either isolated GST isoenzymes or mixtures thereof which
contain known compositions or both. The specifically reactive reagents
must include, as a panel, at least one, preferably three, and more
preferably five GSH analogs of formula 1, along with additional reagents,
if desired. Such additional reagents may include a series of known
substrates wherein turnover rates are measured. Suitable substrate
candidates include, for example, ethacrynic acid, bromosulfophthalein,
cumene hydroperoxide, BCNU, chlorambucil, trans-stilbene oxide and the
like.
Also available for use as specifically reacting reagents which are members
of the panel to provide a SC profile are inhibitors which interact with
the GST isoenzyme at various levels. These inhibitors include, for
example, piriprost, Cibacron Blue, and hematin. Antibodies which are
specifically immunoreactive with the various isoenzymes can be used, as
well as paralog-type affinity reagents. If the profile is to provide a
basis for therapeutic strategy design, it is preferred that at least some
members of the panel must be descriptive of the enzymatic activity of the
GST.
An additional technique for obtaining SC profiles is analogous to that
described by Takeo, K., et al., J Immunol (1978) 121:2305-2310. In this
approach, differential electrophoresis in the presence of various binding
agents for the individual proteins permits measurement of a mobility
value. In the specific application described by Takeo, measurements of
dextran-specific myeloma proteins in polyacrylamide gel electrophoresis
were made, showing retardation when the dextrans were added to the
separating gel, which retardation could be reversed by adding the hapten
isomaltose oligosaccharide. In using this approach, a series of mobilities
depending on the choice of retarding agent, for example, could be obtained
for known compositions. This technique may be applied by using the novel
compounds or panels of the invention as the retarding or mobilizing
agents.
In one preferred method for determining the GST complement of biopsies, a
series of HPLC columns is constructed using the known GST substrates
studied by Mannervik, B., et al., Proc Natl Acad Sci (1985) 82:7202-7206.
These substrates are conjugated directly to the column supports or are
attached to the GSH analog variants described by Adang, A. E. P., et al.,
Biochem J (1990) 269:47-54. A series of 50-100 different columns resulting
from the various possible combinations of substrates with GSH analogs of
formula 1 represents a series of candidate sorbents. These sorbents are
tested to select those of maximal diversity in properties by utilizing
each for the separation of a mixture of known GST isoenzymes. The four or
five columns with the greatest differentiation capacities are then chosen
as panel members for determining SC profiles in unknown samples and in
standards.
Thus, rather than displaying the separations as elution patterns on each
individual sorbent, the data are rearranged so that the capacity for
adsorption to each sorbent represents an information channel in the SC
profile of the isoenzyme. The reactivity pattern with respect to inducers,
activators, substrates, and inhibitors are also determined for each
isoenzyme and used as an information channel. The completed profile for
each known isoenzyme is then used as a member of a reference set.
Additional members of the reference sets are determined by utilizing
samples from normal tissues and evaluating the values assigned to the same
set of information channels. The corresponding profiles of biopsy samples
from unknown, unwanted tissues are then compared against this reference
set.
The profiles for known compositions are stored in computationally
accessible form for comparison to profiles similarly determined for
unknown samples. Thus, kits can be provided for determination of the GST
complement of unknown samples which include the test panel members used in
determination of the reference profiles along with instructions for SC
profile determination of the unknowns. Suitable software to access the
reference profiles may also be included. The GST complement can be used to
characterize the sample tested and, if appropriate, may be used to design
therapy.
For diseased tissue, the appropriate strategy can be selected for
treatment. The complement may be evaluated to determine whether or not
standard treatment protocols will be successful when applied to the
unwanted cells or tissues or may be used for the design of different
protocols including the choice of toxin or prodrug and the inclusion or
noninclusion of a therapeutic modulator.
Svnthesis of the Novel Tripeptide Analogs
The novel tripeptide analogs of the invention or additional tripeptide
analog members of diverse panels can be synthesized using means generally
known in the art, but using modifications which render these general
methods applicable to the desired tripeptide. Although solid-phase
synthesis methodologies can be used, liquid-phase peptide synthesis
appears superior for these short peptides. The Fmoc reagents originally
developed for solid-phase synthesis can be applied to a solution-phase
method for producing 100 mg quantities of the tripeptide analogs.
The intermediate protected dipeptides and tripeptides synthesized using
solution-phase Fmoc chemistry are isolated by chromatography on silica
gel, and deprotected in mild base, thus allowing synthesis of acid-labile
thioconjugates (Iselin, B., et al., Helv Chem Acta (1955) 38:1508-1516).
The analogs can be purified and recovered, or the crude product mixtures
may be directly coupled to solid support to provide affinity-derivatized
supports (Sundburg, L., et al., J Chromatog (1974) 90:87-98).
In those circumstances where ester of the C-terminal amino acid AA.sub.C is
not available, the ester is made by synthesizing the N-Fmoc-protected
amino acid (Atherton, E., et al., in "Solid Phase Peptide Synthesis," IRL
Press, Oxford, England (1989), pages 47-61) and then esterified by
treatment with the desired alcohol in the presence of concentrated
sulfuric acid (Benoiton, L., Can J Chem (1962) 40:570-572). Nonesterified
materials are removed by extractions with mild base and the desired N-Fmoc
amino acid ester is isolated by evaporation.
The sulfur-functionalized Fmoc cysteine derivatives are made in a one-pot
procedure by treating cysteine with Fmoc-OSu as pH 9 and then treating
this mixture with the appropriate alkylating agent.
The synthesis is conducted as shown in Reaction Scheme 1.
##STR5##
Coupling the cysteine derivative to the C-terminal amino acid is
accomplished with the water-soluble carbodiimide EDC (Sheehan, J., et al.,
J Org Chem (1961) 26:2525-2528) and HOBT (Konig, W., et al., Chem Ber
(1970) 103:788-798) (added to retard undesired racemization and speed up
the reaction) in DMF. After coupling is complete, usually about 1 hr at
r.t., the mixture is reduced in vacuo and poured into KHCO.sub.3 solution
(John Hughes, private communication). This step extracts most of the DMF
and EDC and EDC urea, as well as some of the Fmoc-cysteine derivative
which did not couple. The resulting gummy residue is retained by decanting
off the liquid. This crude dipeptide is dissolved in EtOAc and washed with
1N hydrochloric acid and water to remove any remaining uncoupled
C-terminal amino acid ester, as well as residual EDC and EDC urea. The
solution is concentrated and the dipeptide purified by chromatography.
The recovered dipeptide is then treated with 25% piperidine in DMF for 30
min to remove the Fmoc group. The dibenzofulvene or its piperidine adduct
resulting from Fmoc removal should not affect the results of the next
coupling (Atherton, E., et al., J Chem Soc Perkin Trans (1981) 1:538-546).
Any excess piperidine, however, must be removed, which is accomplished by
repeated co-evaporation with DMF until the odor of piperidine is no longer
detectable in the deblocked material. The second coupling is then
performed with the glutamic acid derivative followed by the same workup as
for the dipeptide.
Fmoc glutamic acid .alpha.-benzyl ester is made in good yield and purity
from commercially available glutamic acid .alpha.-benzyl ester.
Fmoc-glutamic acid .alpha.-tert butyl ester, also commercially available,
can also be used, but this requires a separate acid treatment step in the
workup. There are solubility problems with some of the protected
tripeptides during this step, and impure, partially deprotected products
are often obtained.
The material produced by the coupling of the glutamic acid derivative to
the dipeptide and workup contains several chromatographically mobile
components. The material that elutes first from the final column is
fluorescent, suggesting dibenzofulvene. The putative desired product
elutes next along with another UV-absorbing material, which is probably
the piperidine adduct of dibenzofulvene. Since similar products are
generated and separated from the deblocked tripeptide during the final
workup, these contaminants are not removed at this stage.
Once the protected tripeptide (and impurities) are isolated, it is dried
under vacuum and treated with 0.25N NaOH in 3:1 ethanol:water for 18 hrs.
This removes the Fmoc and ester-protecting groups. When t-butyl-protected
glutamic acid is used, 3N HCl in ethanol/water 3/1 v/v for 3 hr is used to
remove the t-butyl group before the base treatment. The acid is removed by
rotary evaporation and co-evaporation with ethanol and water, and the same
base treatment as above removes the remaining protecting groups. After the
overnight base treatment, addition of water and extraction with hexane
removes the organic by-products of the deprotection. The aqueous solution
of the peptide is acidified and reduced to a solid. Dissolution of the
peptide in ethanol and filtration removes the salt. This is evaporated to
a foam and subjected to high vacuum overnight.
The compounds are analyzed by HPLC, TLC and FAB mass spectroscopy. While
the TLC analysis show good results in most cases, the HPLC results are
mixed, partially because the analysis conditions used were not optimized
for some of the more hydrophobic (S-alkyl C-terminal valine,
.beta.-alanine and 4ABu) peptides.
Racemization during the final deprotection with base may occur in some
small amount, particularly with the phenylglycine analog (Bodansky, M., et
al., in "Practical Methods in Peptide Synthesis," Springer Verlag, Berlin
(1984)). This is less than that which occurs with the sodium-ammonia
conditions used previously by Adang, A. et al. (Biochem J (1989)
264:721-724).
Using the techniques set forth above, the analogs of Table 1 were prepared.
TABLE 1
______________________________________
Compound.sup.a
Yield, %.sup.b
TLC R.sub.f.sup.c
M/e.sup.d Loading .sup.e
______________________________________
.gamma.E-C(Bz)-G
32 0.49 388.2, 402.2.sup.f
1.0
.gamma.E-C(Pr)-A
23 0.71 365.2 9.0
.gamma.E-C(Hx)-A
17 0.76 406.2, 428.2.sup.f
4.8
.gamma.E-C(Bz)-A
44 0.35 412.2, 434.2.sup.f
6.6
.gamma.E-C(Trt)-A
15 0.83 586.4.sup.f
1.2
.gamma.E-C(Me)-.beta.A
27 0.58 357.1.sup.f
.gamma.E-C(Pr)-.beta.A
27 0.41 364.1, 386.1.sup.f
.gamma.E-C(Hx)-.beta.A
13 0.49 406.3, 428.3.sup.f
6.0
.gamma.E-C(Bz)-.beta.A
17 0.66 434.2.sup.f
8.1
.gamma.E-C(Trt)-.beta.A
42 0.92 564.3, 586.5.sup.f
N/A
.gamma.E-C(Pr)-4ABu
13 0.51 378, 400.sup.f
.gamma.E-C(Hx)-4ABu
17 0.52 402.3, 424.3.sup.g
4.6
.gamma.E-C(Bz)-4ABu
23 0.70 426, 448.2.sup.f
4.0
.gamma.E-C(Pr)-V
23 0.67 391 13.7
.gamma.E-C(Hx)-V
15 0.64 434.2, 456.2.sup.f
19.5
.gamma.E-C(Bz)-V
26 0.73 440.1, 462.1.sup.f
6.5
.gamma.E-C(Pr)-V
33 0.55 408 7.0
.gamma.E-C(Hx)-D
25 0.68 451.2 5.9
.gamma.E-C(Bz)-D
22 0.59 456.1, 478.1.sup.f
2.4
.gamma.E-C(Pr)-PG
14 0.64 426.4, 448.sup.f
.gamma.E-C(Hx)-PG
13 0.63 468.3 4.8
.gamma.E-C(Bz)-PG
11 0.61 474.1, 496.1.sup.f
2.0
.gamma.E-C(Pr)-H
6 0.57 429.2
.gamma.E-C(Hx)-H
30 0.61 473.3 6.0
.gamma.E-C(Bz)-H
11 0.58 499.3.sup.f
1.0
______________________________________
.sup.a Standard oneletter AA code, with Me = methyl, Pr = npropyl, Hx =
nhexyl, Bz = benzyl, Trt = trityl (triphenyl methyl).
.sup.b Moles of deprotected product divided by the moles of Fmoccysteine
derivative used.
.sup.c TLC R.sub.f values for silica plates eluted with
EtOAc/pyridine/HOAc/water 5/5/3/1 and visualized with ninhydrin spray.
.sup.d Observed molecular mass, in AMU, usually the molecular weight plus
1. Thioglycerol or nitrobenzyl alcohol matrix.
.sup.e Micromoles of peptide per mL of swollen resin volume (water)
.sup.f Sodium adduct, M + 22.
.sup.g Molecular ion and sodium adduct minus water; MH.sup.+ 18.
In addition, other compounds of formula 1, including the cycloamido forms
of these compounds, as well as the ethyl and benzyl esters thereof, are
prepared.
The following examples are intended to illustrate, but not to limit, the
invention.
EXAMPLE 1
Synthesis of 9-Fluorenylmethoxycarbonyl-4-aminobutyric acid ethyl ester
Forty-five g (0.1339M, 0.94 eq) of Fmoc-Osu was added slowly to a solution
of 14.75 g (0.143M, 1 eq) of 4-aminobutyric acid (4-ABu) and 20 g of
Na.sub.2 CO.sub.3 in 300 mL of deionized water and 200 mL of
tetrahydrofuran (THF). The pH was monitored and more Na.sub.2 CO.sub.3 was
added to keep the pH above 8. The reaction was stirred for 2 hr and then
acidified with conc. HCl. The resulting cloudy suspension was extracted
with 600 mL of ethyl acetate (EtOAc), after which the organic layer was
further extracted with 300 mL 0.5N NaOH. The aqueous layer was rapidly
poured into 20 mL of conc. HCl in 500 mL of ice water. The resulting white
suspension was extracted with 300 mL of EtOAc, dried over 50 g of Na.sub.2
SO.sub.4 and evaporated to 35 g (76% yield) of Fmoc-4-ABu as a white
powder. This was dissolved in 500 mL of absolute ethanol and 40 mL of
conc. H.sub.2 SO.sub.4 was added. After 4 hrs, the solution had become a
semi-solid white mass. This was poured into 2 L of water and filtered. The
white material was dissolved in 500 mL of EtOAc and extracted once with
200 mL of 0.5N NaOH, dried and evaporated to give 30 g (79% yield) of
total compound, R.sub.f 0.71 (20% MeOH in CH.sub.2 Cl.sub.2), mp
83.degree.-86.degree..
Anal. Calcd. for C.sub.21 H.sub.23 NO.sub.4 : C, 71.37; H, 6.56; N, 3.96.
Found: C, 71.42; H, 6.67; N, 3.72.
EXAMPLE 2
Synthesis of 9-flourenylmethoxycarbonylphenylalycine ethyl ester
In a similar synthetic procedure, 20 g of phenylglycine gave 33.7 g (68%
yield) of Fmoc-phenylglycine. 19 g of this product was converted into 10 g
(57% yield) of product, Rf 0.95 (same TLC system), mp
130.degree.-133.degree..
Anal. calcd. for C.sub.25 H.sub.23 NO.sub.4 : C, 74.80. H, 5.77. N, 3.49.
Found: C, 74.72, H, 5.91, N, 3.20.
EXAMPLE 3
Synthesis of 9-flourenylmethoxycarbonyl-aspartic acid dimethyl ester
Forty-five g (0.134M, 0.96 eq) of Fmoc-Osu was added to 20 g (0.15M, 1 eq)
of aspartic acid and 20 g of Na.sub.2 CO.sub.3 dissolved in 400 mL of
water and 200 mL of dioxane. The mixture was stirred for 2 hrs while the
pH was maintained at about 9 by the addition of small amounts of Na.sub.2
CO.sub.3. Then the cloudy white mixture was poured into 500 mL of ice
water containing 40 mL of conc HCl. The white solid was extracted with 500
mL EtOAc and this was mixed with 500 mL of hexane. The mixture was chilled
overnight and stirred the next day to give 38 g of Fmoc-aspartic acid as
crystals (71% yield) upon filtration and air drying. 10 g (0.28M) of this
product was dissolved in 200 mL of methanol and 20 mL of conc H.sub.2
SO.sub.4 was added. The solution was allowed to stand overnight. The
mixture was poured into 1 L of water and filtered. The resulting white
solid was dried and redissolved in EtOAc. Slow addition of hexane and
chilling gave 9 g (83% yield) of product as white needles, mp
78.degree.-80.degree.. ›a!.sub.d =-13.9.degree..
Anal. calcd. for C.sub.2 H.sub.21 NO.sub.4 : C, 65.78. H, 5.52. N, 3.65.
Found: C, 66.18. H, 5.68. N, 3.69.
EXAMPLE 4
Synthesis of 9-flourenylmethoxycarbonyl-S-hexyl cysteine
A. Twenty g (0.127M, 1 eq) of cysteine hydrochloride and 20 g Na.sub.2
CO.sub.3 was dissolved in 800 ml of water under a stream of argon. Two
hundred mL of CH.sub.3 CN was added, and then 42 g (0.122M, 0.96 eq) of
Fmoc-Osu was added in small portions while the pH was maintained at about
9 by adding 5 g portions of Na.sub.2 CO.sub.3. The reaction was stirred
for an additional 2 hrs, and 18.6 mL (26.8 g, 0.126M, 0.99 eq) of
1-iodohexane was added as a solution in 200 mL of CH.sub.3 CN. The
reaction was stirred for an additional 2 hrs and poured into 1 L of ice
water and 50 mL of conc HCl. The white mixture was extracted with 600 mL
of EtOAc, and the organic layer was extracted with 2 500 mL portions of 1N
KOH. Each of these was immediately dropped into separate portions of 500
ml of water and 30 mL of conc HCL, and the cloudy mixtures obtained were
each extracted with 500 ml of EtOAc. These were each dried over Na.sub.2
SO.sub.4 and evaporated. The total yield was 24.6 g (45%). The second
fraction (3.5 g) crystallized on standing, mp 10.degree.-103.degree..
R.sub.f =0.57. ›a!.sub.d =-14.3.degree..
Anal. Calcd. for C.sub.21 H.sub.23 NO.sub.4 S: C, 65.42. H, 6.01. N, 3.63.
Found: C, 65.53. H, 5.74. N, 2.91.
B. Additional S-functionalized Fmoc cysteine derivatives were prepared as
set forth in paragraph A.
EXAMPLE 5
Synthesis of Fmoc-glutamic acid .alpha.-benzyl ester
Twenty-five g (0.105M) glutamic acid .alpha.-benzyl ester and 25 g Na.sub.2
CO.sub.4 was dissolved in 400 mL of water and 200 mL THF was added. 34 g
(0.101M, 0.96 eq) Fmoc-OSu was added in small portions with stirring, and
the pH was kept at about 9 by adding more Na.sub.2 CO.sub.3 as needed.
After 1 hr, the reaction was poured into 500 mL of water and acidified
with conc HCl. The white suspension was extracted with EtOAc, dried over
Na.sub.2 SO.sub.4 and evaporated to a solid mass. This was dissolved in
500 mL hot EtOAc and 300 mL hexane was added. Overnight chilling,
collection and air-drying gave 38.7 g (83% yield) of white crystals, mp
110.degree.-112.degree.. ›a!.sub.d =-13.8.degree.. M/e (Rel. inten.):
460.2 (19), 363.4 (8), 345.4 (19), 307.2 (10), 289.2 (12), 238.2 (12),
191.2 (10), 178.2 (89), 165.1 (23), 154.1 (57), 136.1 (48). .sup.1 H NMR
(400 mH z), PPM: 1.9 (m, 1H ), 2.2 (m, 1H ), 2.4 (M, 2H ), 4.1 (t, 1H ),
4.4 (d, 2H ), 4.43 (m, 1H ), 5.1 (s, 2H ), 5.6 (d, 1H ), 7.3 (m, 9H ), 7.5
(d, 2H ), 7.7 (d, 2H ), 9.4-9.6 (broad s, 1H ). .sup.13 C (100 mHz), PPM:
27.5, 30.0, 47.3, 53.5, 67.3, 67.7, 120.2, 125.0, 127.3, 128.5, 128.8,
129.2, 135.2, 141.5, 143.6, 143.9, 156.3, 171.4, 177.8.
Anal. Calcd. for C.sub.27 H.sub.25 NO.sub.6 : C, 70.57. H, 5.48. N, 3.05.
Found: C, 69.71. H, 5.58. N, 2.88.
EXAMPLE 6
Synthesis of .gamma.-glutamyl S-benzyl cysteinyl .beta.-alanine
1.5 g (9.76 mmol, 1 eq) of .beta.-alanine ethyl ester hydrochloride was
added to 50 mL of DMF and 1.8 mL of DIPEA was added. 3.5 g (8.1 mM, 0.83
eq) of Fmoc-S-benzyl cysteine was added and dissolved by swirling the
solution. Next 2 g of EDAC and 250 mg of HOBT were added, and the solids
were dissolved by swirling. The mixture was allowed to stand for 1 hr, and
was concentrated in vacuo to a mobile oil of about 5 mL in volume. To this
was added 100 mL of 10% weight/volume KHCO.sub.3 in water, and the mixture
was shaken and the liquid removed by filtration. The residue was dissolved
in 100 mL of EtOAc, washed with 50 mL of 1N HCl, 50 mL of water and dried
over Na.sub.2 SO.sub.4. The solution was filtered and concentrated in
vacuo to give a foam which was chromatographed using a 2.times.6 cm bed of
silica gel packed in CH.sub.2 Cl.sub.2. The column was eluted until the
first UV absorbing material appeared, then a gradient was run in 1%
methanol increments of 100 mL volume to 3% methanol. A strong UV absorbing
band eluted after two portions of 3% methanol; these were checked for
purity by TLC and pooled and evaporated in vacuo to give 4.6 g (83% yield)
of Fmoc-Cys(S-benzyl)-.beta.-alanine ethyl ester. Half of this (4 mmol)
was dissolved in a mixture of 30 mL DMF and 10 mL piperidine, and allowed
to stand for 30 min. The solution was reduced to a solid in vacuo, and the
process was repeated twice again with 50 mL of DMF. The resulting white
solid was subjected to a high vacuum for 1 hr, then it was dissolved in 50
mL of DMF. For the second coupling step, 1.6 g (3.5 mmol, 0.9 eq) of
Fmoc-glutamic acid (.alpha.) benzyl ester (v) was added, followed by 0.8 g
(4.2 mmol, 1.05 eq) of EDAC and 200 mg (1.4 mmol) of HOBT. The mixture was
allowed to stand 1 hr, and concentrated in vacuo to about 5 mL in volume.
This was poured into 100 mL of 10% aqueous KHCO.sub.3 solution and shaken.
The liquid was poured off, and the residue was dissolved in 100 mL of
ethyl acetate. The organic layer was washed with 50 mL of 1N HCl, 50 mL of
water, and dried over Na.sub.2 SO.sub.4. This was reduced to a tar and
chromatographed in the same manner as the protected dipeptide. 1.2 g (42%
yield) of the protected tripeptide was obtained. This material was
dissolved in 30 mL of absolute ethanol and 10 mL of 1N NaOH solution was
added. The mixture was allowed to stand for 18 hrs, and was poured into a
separatory funnel with 40 mL of water and 40 mL of hexane. The layers were
shaken and separated, and the aqueous phase was washed with an additional
40 mL of hexane. The pH of the water layer was adjusted to about 3 by
adding a few drops of conc HCl, and the cloudy solution was reduced to a
solid in vacuo and subjected to a high vacuum for several hrs. The residue
was washed with 2 20 mL portions of absolute ethanol. The ethanol-NaCl
slurry was filtered, and the clear solution was evaporated to a tar. The
weight was 620 mg (89% yield from the protected tripeptide, 19% from
Fmoc-Cys(Benzyl)-OH). A TLC plate was run in ethyl
acetate/pyridine/water/acetic acid 5/5/3/1 v/v and visualized with
ninhydrin spray and heat (Stewart, J. et al., "Solid Phase Peptide
Synthesis" (1984) pp. 53-124, Pierce Chemical Co., Rockford, Ill, R.sub.f
0.66. HPLC analysis showed 74% purity by area integration of UV absorbing
material (FIG. 1). Fast atom bombardment Mass spectroscopy (FABMS) showed
an ion peak at 434.2 M/e, consistent with the tripeptide monosodium salt.
Other higher mass peaks were also present, attributable in part to
incompletely deprotected peptide.
In cases where the Fmoc protected C-terminal amino acid ester was used
instead of the commercially available free amino C-terminal amino acid
ester, this material was deblocked with the same procedures as those used
to deblock the protected dipeptide above, then proceeding with the first
coupling reaction as above.
EXAMPLE 7
Derivatization to Sepharose Resin
0.66 g epoxy Sepharose.TM. 6B (Pharmacia) was swollen with 10 mL water for
15 min, then rinsed twice with 10 mL water in a 15 mL sintered glass
funnel. A solution of 100-500 mg of the crude tripeptide in 5 mL ethanol
and 10 mL water was adjusted to pH 1-12 with 6N NaOH in a 20 mL
scintillation vial with a polycone cap. The rinsed resin was added, and
gently agitated overnight in a 37.degree. water bath. The pH was checked
the next day and brought back to pH 11-12 with 6N NaOH if needed. After
another day of agitation, the resin was filtered (the peptide-containing
liquid was acidified with conc HCl, evaporated and saved) and rinsed three
times with 10 mL water. The unfunctionalized epoxy groups were capped by
soaking the resin in 10 mL of water which contained about 0.1 mL
ethanolamine for 1 hr. The resin was then rinsed three times with 10 mL
water, and a sample was removed for analysis. The remainder was rinsed
with 10 mL of 0.1M NaOAc, 0.5M NaCl pH 4.0 buffer, followed by 10 mL of
0.1M tris chloride, 0.5 M NaCl pH 8.0 buffer. The resin was stored at
4.degree. C. in this pH 8 buffer.
EXAMPLE 8
Use of the Compounds of the Invention as Affinity Sorbents
A series of the compounds of formula 1 was constructed wherein YCO was
.gamma.-Glu, AA.sub.C was Gly, and X was benzyl having at the para
position, nitro, chloro, methoxy and methyl substituents. The relevant
analogs were derivatized to Sepharose resin as described above and used as
affinity sorbents in the separation of three recombinant human GST
enzymes. HPLC was used to measure the relative amounts of the enzymes
which bound to the supports.
The results are shown in Table 2.
TABLE 2
______________________________________
Substituent % .pi. % .mu.
______________________________________
NO2 95 5
Cl 89 11
OMe 70 17
Me 3 94
______________________________________
When the percentage of .pi. and .mu. isoenzymes were plotted against their
sigma (meta) values good linear correlation was obtained. However, poor
correlation was obtained when the sigma (para) values were used. The sigma
(meta) values are a measure of purely inductive effect; however, the sigma
(para) values are dependent on resonance in which the substituent can
place partial charges at an atom in the ring next to the point of
attachment.
The recoveries of protein and GST (CDNB-conjugating) activity in the
elution fractions after affinity purification of rat liver cytosolic GST
isoenzymes on various compounds of the invention are shown in Table 3.
TABLE 3
______________________________________
Recovery of protein and CDNB-conjugating activity after
affinity chromatography of rat liver cytosol
on GSH and selected GSH analog sorbents
Protein Activity
Sorbent (.mu.g)
(% total) (.mu.mol/min/ml)
(% total)
______________________________________
HxGSH 60 3.0 1.32 67
S-L-GSH 51 2.5 1.21 61
.gamma.E-C(Cu)-G
49 2.5 0.64 32
.gamma.E-C(Bz).beta.A
20 1.0 0.69 35
.gamma.E-C(Bz)-A
11 0.5 0.43 22
.gamma.E-C(Hx)-.phi.G
4 0.2 0.10 5
______________________________________
Abbreviation: Cu = cumyl (.alpha.,dimethylbenzyl)
Traditional S-L-GSH and HxGSH sorbents showed high binding and broad
isoenzyme recognition. The novel sorbents retained a lower mass of the GST
isoenzymes, with the protein recovery for three of the novel sorbents
being less than half that of the traditional sorbents.
In comparison with HxGSH, the novel sorbents did not show a direct
correspondence of CDNB-conjugating activity with protein recovery. For
example, .gamma.E-C(Bz)-.beta.A and .gamma.E-C(Bz)-A sorbents gave 33% and
16% of the protein recovery seen with HxGSH, but the CDNB-conjugating
activities were far higher (52% and 33% respectively) after correcting for
the inhibitory properties of the eluting ligand. In contrast, protein
recovery by .gamma.E-C(Cu)-G sorbent decreased minimally, while activity
decreased by 50%. It is well known that different GST isoenzymes display
individual specific activities with CDNB. The discrepancy between protein
recovery and activity recovery was attributed to differences in the
isoenzyme composition in the elution fractions of each sorbent. This
hypothesis was supported by SDS/PAGE and reverse-phase HPLC analysis of
the eluate fractions.
Fractions eluted from the sorbents listed in Table 3 were analyzed by
SDS/PAGE followed by silver staining. S-L-GSH and HxGSH sorbents bound
proteins which corresponded to the previously observed electrophoretic
band pattern of rat liver GST subunits. The novel sorbents displayed
different band patterns. For example, the .gamma.E-C(Bz)-.gamma.A eluate
referred to above showed a highly enriched band for presumptive .mu.-class
isoenzymes. As the .pi.-class GST 7-7 is found at low levels in rat liver
cytosol, other rat tissues known to express higher levels of this
isoenzyme also were tested. The GST gel pattern from rat kidney cytosolic
proteins eluted from the .gamma.E-C(Hx)-.phi.G sorbent showed a protein
with the same molecular migration as rat GST subunit 7 is the unique
component of this eluate, indicating that this sorbent is a .pi.-selective
reagent.
A protein with a molecular migration similar to that of another
GSH-dependent enzyme, glyoxalase I, was also present in the eluates from
.gamma.E-C(Bz)-A and .gamma.E-C(Bz)-.beta.A, but was not detected in
eluates from the other sorbents. In these experiments HxGSH, previously
shown to bind glyoxalase I, did not show any detectable levels of this
GSH-dependent enzyme. Glyoxalase I is known to be a metal-dependent
enzyme, and the presence of EDTA in the buffers may have interfered with
binding activity.
The high degree of purity of appropriately sized proteins seen by SDS/PAGE
of the affinity-eluted proteins suggested that the observed bands are
indeed GSTs. Independent confirmation of this conclusion, at higher
resolution, was provided by reverse-phase HPLC analyses of the GST
isoenzymes in the eluates from different sorbents. Comparison with
purified GST standards showed that the isoenzymes eluted from the novel
sorbents correspond to known GST isoenzymes. These procedures showed that
GST subunit 3 eluted at 14 min, subunit 4 at 17 min, subunit 7 at 18 min,
subunit 2 at 23 min, subunit 6 at 25 min, subunits 1a and 1b at 37 and 40
min, and subunit 8 at 42 min. Subunits 3, 4 and 6 are .mu.-class
isoenzymes, subunits 1a, 1b, 2 and 8 are .alpha.-class isoenzymes, and
subunit 7 is the .pi.-class isoenzyme. S-L-GSH and HxGSH sorbents, as
expected, bound all except 5-5, the Theta-class isoenzyme.
.gamma.E-C(Cu)-G increased the proportion of isoenzyme 2, yet bound all the
isoenzymes to some extent. In contrast, other sorbents were very selective
in their binding of the isoenzymes. For example, .gamma.E-C(Bz)-.beta.A
selectively bound the .mu.-class isoenzymes 3 and 4, confirming the
SDS/PAGE results.
The specificities of a variety of the novel sorbents for rat cytosolic GST
isoenzymes are summarized in Table 4. Some structural alterations of the
ligand produced sorbents that did not effectively bind any isoenzyme;
several of these are listed for completeness.
TABLE 4
______________________________________
Separation of rat GST isoenzymes by novel sorbents
Isoenzyme Protein
subunit recovery
Sorbent* specificity (%)
______________________________________
High binding, nonselective
Hx-GSH All 3.0
S-L-GSH All 2.5
Selective
.gamma.E-C(Cu)-G
2 > 3 > 4 2.5
.gamma.E-C(BuBz)-G
2 > 4 > 3 > 7 1.7
.gamma.E-C(Bz)-A
4 > 3 >> 6 > 2
0.5
.gamma.E-C(Bz)-.beta.A
3 = 4 >> 2 > 6
1.0
.gamma.E-C(Pr)-H
4 > 3 >> 2 1.0
.gamma.E-C(Pr)-A
3 = 4 > 2 > 1 > 6 > 7
1.4
.gamma.E-C(Hx)-.phi.G
7 >> 3 = 4 0.2
.gamma.E-C(Bz)-.phi.G
7 >> 2 = 3 = 4
1.3
Weak binding
.gamma.E-C(Pr)-V
Little: 2 = 3 = 4
0.5
.gamma.E-C(Hx)-V
Little: 2 - 3 - 4
0.4
.gamma.E-C(Pr)-D
Little: 3 > 2 > 4
0.2
.gamma.E-C(Hx)-D
None 0.1
.gamma.E-C(Bz)-H
None 0.1
______________________________________
*Abbreviations as in Table 1 and text above, except Cu = Cumyl
(.alpha.,dimethylbenzyl) and .phi.G = phenylglycine.
Systematic alterations of the Cys residue to probe the previously defined
H-site (hydrophobic pocket) resulted in ligands capable of binding to the
isoenzymes. Adang et al. (1990) did not examine any analogues with
systematic modifications of groups attached to the Cys moiety. Alterations
of this group had effects on the level of protein recovery and the
specificity of isoenzyme binding in several cases. For instance,
alteration of the S-linked moiety of glutathione to a structure similar to
that of cumene hydroperoxide, an .alpha.-class substrate, made the
sorbents preferential for isoenzyme 2-2 of the .alpha.-class. When Ala was
used as the terminal amino acid, both propyl and benzyl moieties on the
Cys resulted in sorbents specific for .mu.-class isoenzymes (Table 4). The
propyl sorbent retained more GST protein, in part because it picks up both
isoenzymes 3 and 4, compared with the benzyl sorbent, which is highly
specific for isoenzyme 4. When His was used as the terminal amino acid,
the propyl moiety resulted in high .mu.-class specificity, while the
benzyl moiety resulted in no binding at all. In contrast to these
sorbents, alterations of the Cys moiety of Val- and Asp-substituted
sorbents had no effect on the low binding. Thus, the Cys substituent can
be important in modifying the overall specificity of the sorbent as
determined by the terminal amino acid.
EXAMPLE 9
Use of the Compounds of the Invention as Solution Phase Inhibitors
Screening Strategy
A systematically diversified set of peptide analogs of the invention has
been tested as isozyme-specific inhibitors of human
glutathione-S-transferase (GST). The potency of the best of the inhibitors
is in the 0.5 to 20 micromolar range, with kinetics indicative of
competitive inhibition with glutathione at the active site. The
specificity observed among three recombinant-derived GST isozymes at both
low and high potency ranged from negligible to high (at least 20-fold over
the next most sensitive isozyme).
These compounds were initially screened at 1-mM concentrations against
three recombinant human GSTs, one from each major isoenzyme class, to see
if they would inhibit the enzyme activity, using CDNB as a
spectrophotometrically detectable substrate. To permit the rapid testing
of a wide spectrum of compounds, the paralogs used at this stage were
crude synthetic samples and were not characterized for purity before
testing, although in most cases the intended synthetic compound was the
dominant species, representing about 50% of the mass. The nominal 1-mM
concentration was based on an assumption of 100% purity. Table 5 shows the
results of these inhibition tests for a series of 51 paralog analogs of
GSH.
TABLE 5
__________________________________________________________________________
Inhibition of human GST activity by GSH analogs
% Inhibition at 1 mM
.mu.M Conc. for IC.sub.50
No.
Structure P1-1
A1-1
M1a-1a
P1-1
A1-1
M1a-1a
__________________________________________________________________________
Peptide analogs of GSH showing inhibition bias toward P1-1:
1 .gamma.E-C(Hx) (IMe)-.phi.G
99 96 83 35 36 450
2 .gamma.E-C(p-ClBz)-.phi.G
99 92 89
3 .gamma.E-C(octyl)-.phi.G
96 92 90 100 1000
300
4 .gamma.E-C(Bz)-.phi.G
85 68 30
5 .gamma.E-C(Hx)-R(-).phi.G
75 62 4
6 .gamma.E-C(Hx)-S(+).phi.G
71 61 48 4 12 100
7 .gamma.E-C(decyl)-.phi.G
70 60 60 300 300 500
8 .gamma.E-.alpha.-aminooctanoic-G
52 1 8
Peptide analogs of GSH showing inhibition bias toward A1-1:
9 .gamma.E-C(octyl)-G
99 99 99 7 1 1
10 .gamma.E-C(Hx)-G
93 99 99 75 25 40
11 .gamma.E-C(p-NO.sub.2 Bz)-.phi.G
90 99 99
12 .gamma.E-C(octyl) (IMe)-G
90 99 99 50 20 50
13 .gamma.E-C(Bz)-4ABu
70 70 95 575 575 570
14 G-.gamma.E-C(octyl)-G
99 91 90 500 100 100
15 cyclic .gamma.E-C(Hx)-.phi.G
83 85 80
16 .gamma.E-C(Bz)-4ABu
4 78 44
17 .gamma.E-C(2,5-dichloroBz)-G
50 70 90 1150
570 570
18 .gamma.E-C(Bz)-V
35 64 74
19 .gamma.E-C(Bz)-D
12 62 57 900 900
20 .gamma.E-C(Hx)-4ABu
25 58 83 3000
875 1750
21 .gamma.E-C(Hx)-V
24 48 47
22 .gamma.E-C(Hx)-D
0 35 30 20000
2000
2000
23 .gamma.E-C(Pr)-H
2 17 3
24 .gamma.E-C(Hx)-H
8 11 1
Peptide analogs of GSH showing inhibition bias toward M1a-1a:
25 .gamma.E-C(p-ClBz)-G
85 98 99 200 20 9
26 .alpha.E-C(Hx)-.phi.G
78 80 99
27 .gamma.E-C(p-MeBz)-G
70 90 99 500 150 40
28 .gamma.E-C(Hx)-.beta.A
8 69 99
29 .gamma.E-C(p-MeBz)-.beta.A
55 65 99 800 600 20
30 .gamma.E-C(Bz)-.beta.A
0 25 99 1000
1000
25
31 cyclic .gamma.E-C(Bz)-.phi.G
11 36 95 6000
1500
70
32 .gamma.E-C(3,5-difluoroBz)-G
70 70 95 575 575 570
33 .gamma.E-C(Bu)-G
91 865 93 48 106 21
34 .gamma.E-C(Trt)-A
46 30 93
35 .gamma.E-C(octyl)-.beta.A
30 60 90 2200
1100
500
36 .gamma.E-C(p-cyanoBz)-G
80 70 90 250 550 500
37 .gamma.E-C(t-Bu)-G
88 74 90
38 .gamma.E-C(p-BrBz)-G
60 50 90 1000
1140
300
39 .gamma.E-C(Bz)-G
26 53 84
40 .gamma.E-C(p-t-BuBz)-G
49 51 82 2000
950 200
41 .gamma.E-C(Bz)-A
40 30 80
42 .gamma.E-C(Hx)-A
8 30 80
43 .gamma.E-C(Me-I) (Bz)-.beta.A
10 20 80 2000
2000
30
44 .gamma.E-G-C(Hx)-.phi.G
30 45 7
45 .gamma.E-C(Cu)-G
38 25 74 3000
1500
400
46 .gamma.E-C(Pr)-A
13 46 62 ND 900 800
47 .gamma.E-C(neopentyl)-G
35 36 62 3000
1200
300
48 .gamma.E-C(p-FBz)-G
10 40 60 20000
3800
640
49 .gamma.E-C(Pr)-D
ND 33 49
50 .gamma.E-C(Pr)-V
12 15 28
51 .gamma.E-C(Bz)-H
4 2 8 25000
25000
10000
__________________________________________________________________________
Abbreviations as in Table 1 and text above, except: Conc.=Concentration;
ND=not detected; IC.sub.50 =Inhibitor Concentration causing 50% reduction
in activity; Bu=butyl; t-Bu=tert-butyl; Cu=cumyl
(.alpha.,.alpha.-dimethylbenzyl); IMe=iodomethyl;
.gamma.E-.alpha.-aminooctanoic-G (cmpd 8)=.gamma.E-C(Hx)-G with C(Hx)
replaced by an analog lacking sulfur; G-.gamma.E-C(octyl)-G (cmpd
14)=additional G at N-terminus of .gamma.E-C(octyl)-G; cyclic=N and C
termini the tripeptide are covalently linked (peptide bond);
.gamma.E-G-C(Hx)-.phi.G (cmpd 44)=an additional G is inserted between G
and C in .gamma.EC(Hx)-.phi.G.
Within Table 5 the paralogs are grouped by human GST isoenzyme selectivity,
and within each selectivity class they are ranked by decreasing potency.
Paralogs that showed strong selectivity for the P1-1 and M1a-1a isozymes
were clearly identified in this survey, but no new selective inhibitor for
A1-1 was found beyond those previously described. The criteria set for
proceeding to screening beyond the test of inhibition at the 1-mM
concentration were: inhibitions of >90% on any GST isozyme or a >6-fold
difference in inhibition between an isozyme pair. The IC.sub.50 value for
those paralogs meeting the initial criteria are also presented in Table 5.
A more accurate measure of the binding of paralogs to GST isozymes is
obtained by means of GSH competition experiments to determine a K.sub.i
value. Such tests were performed on those compounds that had IC.sub.50
potencies in the 10- to 100-.mu.M range or exhibited >5-fold selectivities
between isozymes, and for which >90% pure compounds had been made. If the
inhibition by the paralog is competitive, then the K.sub.m values for GSH
in the presence and absence of inhibitor can be used to determine the
K.sub.i value, or dissociation constant, between the paralog and the GST
isozyme. The IC.sub.50 values were used to establish an appropriate
concentration of inhibitor to yield a roughly 50% inhibition at 1 mM GSH.
Data obtained in the GSH competition experiment with various paralogs were
plotted in a Hanes-Woolf plot. Parallel lines of the inhibited and
noninhibited conditions in a Hanes-Woolf plot indicates that the paralog
interacts with the GST-active site. For most combinations of paralog and
GST, strict parallelism was observed. The K.sub.m value obtained for GSH
alone in this entire series of experiments ranged between 0.4 and 0.6 mM,
which was approximately 10-100 orders of magnitude weaker than that
recorded for the most potent paralog.
Table 6 presents the K.sub.i values for this subset of interesting
compounds.
TABLE 6
______________________________________
Inhibition of human GST activity by purified GST paralogs:
micromolar K.sub.i conc. determined by competition binding with GSH
K.sub.i Values
Structure .alpha. .mu.1-1 .mu.2-2
.pi.
______________________________________
.gamma.E-C(Hx)-G
0.84 2.0 36.0 10.0
.gamma.E-C(Hx)-.phi.G
5.8 41.0 97.0 0.85
.gamma.E-C(Hx)-.beta.A
43.0 11.0 42.0 550.0
.gamma.E-C(Bz)-.beta.A
360.0 22.0 26.0 710.0
.gamma.E-C(Bz)-.phi.G
20.0 25.0 31.0 0.45
.gamma.E-C(p-MeBz)-.beta.A
43.0 2.1 20.0 40.0
.gamma.E-C(p-ClBz)-.phi.G
10 11 16 0.09
______________________________________
Most of these compounds appeared more potent in these studies than in the
original screening tests because the new preparations used for the GSH
competition experiments were at least 90% pure. A further contribution
comes from the arithmetic definition of IC.sub.50 as compared with K.sub.i
; the values are equal only as both the GSH and the CDNB concentrations
approach Zero, which was not the case in the present standard protocol.
With regard to specificity, the more accurate determinations were
generally consistent with the preliminary data, although in several cases
relative preferences were changed.
Ouantitative Structure/Activity Relationships
Following standard medicinal chemistry procedures, trends were sought in
the quantitative structure/activity relationship (QSAR) of these
compounds. For this purpose, a series of n-alkyl derivatives of GSH were
tested. Results of a detailed exploration of this feature is summarized in
Table 7.
TABLE 7
______________________________________
Inhibition of GST activity by S-(n-alkyl)-GSH adducts;
micromolar concentration required for 50% inhibition
S-(n-alkvl)-GSH
GST isozyme
C number Name P1-1 A1-1 Mla-la
______________________________________
C1 Methyl 1,000 2,500 1,300
C2 Ethyl 750 1,800 650
C3 Propyl 500 900 100
C4 Butyl 200 140 20
C5 Pentyl 100 10 5.0
C6 Hexyl 80 2.0 3.0
C7 Heptyl 20 1.5 1.5
C8 Octyl 10 1.5 1.5
C9 Nonyl 1.5 1.0 1.0
C10 Decyl 0.75 1.0 0.75
______________________________________
Abbreviation: C = Compound
These results show, first, that the inhibitory potency of the compounds
increases with the chain length of the n-alkyl group and, second, that
this parameter does not generate a regular trend in isozyme selectivity.
It is striking that the results obtained for the paralog compounds
presented in Table 5 show a much greater diversity in the pattern of
inhibition than do those obtained for the set of n-alkyl derivatives of
GSH presented in Table 7.
Numerous small organic molecules have also been previously reported to
inhibit GST activity (Mannervik, B., et al., CRC Crit Rev Biochem (1988)
23:283-337), although only a few have been carefully characterized with
respect to isolated human isozymes. Since comparisons of these compounds
with the radically different peptide-based structures were expected to be
useful for a more detailed QSAR study, testing of such compounds also was
undertaken. Several of these compounds showed potency and selectivity
comparable to those of the peptide-based compounds. The results are
summarized in Table 8.
TABLE 8
______________________________________
Inhibition of GST activity by nonpeptide
(small organic) compounds;
micromolar concentration required for 50% inhibition
GST isozyme
Abbreviation
Compound P1-1 A1-1 Mla-la
______________________________________
cb Cibacron blue 0.4 4.0 1.5
dx Doxorubicin 700 80 500
ea Ethacrynic acid
4.0 2.0 3.0
gs Gossypol acetic acid
200 4.0 4.0
hm Hematin 1,000 2.0 5.0
rb Rose bengal 8.0 0.5 1.0
sp Sulfobromophthalein
20 25 5.0
in Indomethacin >600 300 >600
eb Eosin b 4.0 2.5 2.0
ey Eosin y 3.5 5.0 30
______________________________________
Graphical analysis of inhibition data
The multidimensional inhibition data (each axis representing the extent of
inhibition for one of the recombinant GSTs) were analyzed on an
IBM-compatible personal computer (PC) by the principal-components
algorithm in Pirouette, a multivariate statistical software package
developed for chemical data by Infometrix, Inc. (Seattle, Wash). The
principal components are the eigenvectors of the covariance matrix with
the largest associated eigenvalues. As such, they represent composite
parameters derived from the original axes to satisfy two conditions: (1)
the principal components are independent (orthogonal) dimensions, and (2)
they account for the largest portion of the variance in the data set.
Consequently, projection of data onto a plane defined by principal
components maximizes the clarity of visualization by spreading the data
out along dimensions of maximal scatter. Points that are close together in
such a projection thereof faithfully display correlations in the original
higher-dimensional space. Target values are also plotted that represent
postulated ideal compounds with 100-fold specificity factors and 0.1-.mu.M
K.sub.i values. For this analysis, inhibition potency was defined as log
(1/IC.sub.50), or log 1/K.sub.i) when this more accurate value was
available.
FIG. 1 presents a graphical analysis of IC.sub.50 data from Tables 5, 7 and
8 combined with the K.sub.i data in Table 6. To determine this plot,
inhibition properties were first plotted in a three-dimensional space
whose axes represent the extent of inhibition for each of the three
recombinant isozymes. The principal components of the data distribution
were then determined (Massart, D. L., et al., Chemometrics: a textbook
(1988), Elsevier, Amsterdam). In simple terms, principal components are
composite factors, derived from the original axes, that are mathematically
optimized to display correlations in a multivariate data set by plotting
points along axes chosen to maximize scatter. The plot shown is a
projection of the data onto the plane defined by the second and third
principal components. The first principal component is a dimension largely
representing potency, and scatter in this property is well displayed in
the tables. Choosing the second and third principal components for FIG. 1
highlights the specificity factor, which is believed to be an equally
important aspect of quality. Such a multivariate plot allows convenient
inspection of the quality (both potency and selectivity) of the tested
reagents. Also indicated in the plot are target properties defined as
100-fold specificity and 0.1-.mu.M K.sub.i.
The multivariate analysis of the potencies and selectivities of the
inhibitors that is presented in FIG. 1 highlights several other
interesting features as well. First, it indicates that the benzyl/phegly
paralog (compound 4, Table 5) comes rather close to the goal of a
P1-1-specific inhibitor. Second, the benzyl/.beta.-ala (compound 30),
4-methylbenzyl/.beta.-ala (compound 29), and cyclic benzyl/phegly
(compound 31) paralogs are the most selective for the M1a-1a enzyme but do
not come as close to the goal as achieved for the P1-1 enzyme. Third, no
novel A1-1-selective paralog was discovered in this survey.
The analysis of small organic molecules shows that a variety of potencies
and specificities are also available from this class of chemical
structures. Many of the most effective of such compounds are extremely
hydrophobic dyes, however, and are thus unlikely to be clinically useful
due to high background binding to proteins in general. Although
competition experiments with these inhibitors have not yet been done to
determine directly whether they are competitive inhibitors, work is in
progress to convert them into GSH derivatives with the hope of achieving
potency and selectivity greater than those available from the compounds
described in this paper.
In the design of more effective inhibitors, the data analysis by
multivariate statistics, as summarized in FIG. 1, should be particularly
useful for defining structural trends. Finally, the empirically observed
trends visualized in this way should provide useful insights for
comparison with structural data on GSTs, including studies using X-ray
crystallography, high-resolution nuclear magnetic resonance (NMR), and
site-directed mutagenesis.
Pharmacological Implications
The diversity of GST activity in tumors, both qualitatively and
quantitatively, represents a significant therapeutic opportunity for
targeting existing chemotherapy drugs preferentially to tumors. The same
diversity, however, poses a major challenge to the pharmaceutical chemist
in that potency alone is not sufficient for utility; selectivity among a
group of closely related enzymes is also needed. The paralog approach of
this invention is well suited to such a challenge. As demonstrated in the
present work, crude ligands are useful as initial probes (Table 5).
Reliable assessment of inhibitory properties, however, requires about 50
mg of purified compounds (Table 6). Systematic sampling is thus a very
useful strategy for limiting the characterization work to a reasonable
level.
Furthermore, the range of structures sampled contributes directly to QSAR
and enzyme-structure correlation studies aimed at identifying features
responsible for isozyme selectivity and for potency. For example, all but
one of the most selective inhibitors for the M1a-1a isozymes have
beta-alanine as the C-terminal amino acid in the GSH moiety of the
paralog. Both of the most selective inhibitors for the P1-1 isozyme have
phenylglycine as the C-terminal amino acid of the GSH moiety.
The best inhibitors obtained in this study show the same potency range as
the previously studied inhibitor hexyl GSH, which is the standard ligand
used for affinity purification of GST isozymes. The potency of these
inhibitors is also similar to or better than that of ethacrynic acid,
which is being tested clinically as a drug to potentiate chemotherapy, and
this observation suggests that these novel inhibitors may also be useful
for the potentiation of chemotherapy. For instance, preferential
expression of P1-1 has been reported in a range of tumors, and a
potentiator based on the P1-1 selective inhibitor benzyl/phegly (compound
4) may thus be useful in cancer therapy.
Among the paralogs identified by this screen for which a K.sub.i value had
been determined (Table 6), all showed competitive inhibition with GSH
indicating that they are targeted to the active site of the GST isozymes
for their effect and, thus, that they should affect the activity of GST
against substrates other than CDNB. A variety of alkylating agents are
substrates for GST, and isozymes differ significantly in their specificity
for these substrates. Differential expression of particular isozymes in
individual tumors may therefore account for variability in responsiveness
to treatment. This problem may be overcome by application of appropriate
paralogs of this invention, first, to determine GST isoenzyme complements
of individual tumors, thereby to identify acceptable cytotoxic agents
least susceptible to the predominant GST isozymes of a given tumor and,
second, to provide paralog inhibitors offering the greatest potentiation
of a selected drug by inhibition of whatever GST activity is still present
against that drug.
For inhibition of GST isozyme activities in living cells, for instance to
potentiate cytotoxic drugs, the nhibitors identified in this example
require modifications according to the present invention, to enhance
permeation of the cell membrane, as described above and exemplified in
Example 11, below.
EXAMPLE 10
Use of the Compounds of the Invention in HPLC Affinity Chromatoaraphy of
Human Liver GSTs
This example describes a rapid method for the determination of the
glutathione S-transferase (GST) isoenzymes, for instance, in human liver,
using a new HPLC affinity support. Liver cytosol is injected directly onto
an HPLC column (0.46.times.5 cm) containing a support with a covalently
bound affinity ligand (S-octyl glutathione) specific for the isoenzymes.
Contaminating cytosolic proteins are removed in a washing step. The
isoenzymes are eluted with a linear gradient of a different affinity
ligand in the mobile phase. Coinciding with the affinity ligand gradient,
a salt gradient (0-200 mM sodium chloride) is applied. In this manner the
isoenzymes are fractionated into the enzymatically active homodimers and
heterodimers. The monomeric and dimeric composition of the fractionated
isoenzymes are determined by SDS-PAGE, ELISA and reversed-phase
chromatography. For one liver three Alpha class isoenzyme subunits,
forming three heterodimers and two homodimers, are detected. Five livers
are analyzed and the homodimer A1-1 is found to be the predominant
glutathione S-transferase isoenzyme. Minor amounts of P1 and Mu class
isoenzymes are also detected. This nondenaturing high performance affinity
chromatography method reduces analysis time by a factor of ten when
compared to other affinity analysis methods for the glutathione
S-transferases.
One available chromatographic method for the separation of a range of
glutathione S-transferases (GSTs) in a dimeric, active form is affinity
chromatography using isocratic and/or gradient elution with a counter
ligand. This method combines the purification of the GSTs from the cytosol
by affinity chromatography with a separation of the individual GSTs.
Resolution of GST homodimers and heterodimers has previously been
accomplished using this technique with either S-hexyl glutathione or
glutathione as the affinity ligand bound to agarose. Due to the large
particle diameter and slow flow rates inherent with agarose gels, the
fractionation time for these studies was approximately 25 h (Hayes, J. D.,
et al. (eds.), Glutathione S-Transferases and Drug Resistance, Taylor and
Francis, London, 1989, p.17) which explains why this technique is not used
for the routine analysis of GSTs. An HPLC column containing immobilized
glutathione as the affinity ligand has been described for the preparative
purification of GSTs (LeCreta, F. P., et al., J Chromatog (1988)
434:83-93), but no attempt was made to separate the individual isoenzymes.
This novel HPLC method of this invention uses a stationary phase with a GST
affinity ligand (e.g., S-octyl glutathione) covalently linked to HPLC
particles packed into an analytical HPLC column.
Affinity matrix. The synthesis of the affinity matrix follows the general
procedure of Sundberg and Porath (J Chromatogr (1974) 90:87). HEMA
(hydroxyethyl methacrylate) BIO 1000, 1,4-butanediol diglycidyl ether and
0.6M sodium hydroxide containing 2 mg/ml of sodium borohydride (0.03/1/1,
w/v/v) were mixed overnight. The particles were filtered and washed with
water, ethyl alcohol and acetone. To 700 mg of the dried particles were
added S-octyl glutathione (75 mg) dissolved in 3.5 ml of 0.5M sodium
carbonate. The suspension was mixed for approximately 90 h. After
filtration the particles were washed with (i) 1M sodium chloride, 0.1M
sodium phosphate (pH 9), (ii) 1M sodium chloride, 0.1 sodium acetate (pH
4.5), (iii) water, (iv) ethyl alcohol and (v) acetone.
Packing of HPLC columns. The affinity material (650 mg) was slurried in 20
ml of water and was packed at high pressure into stainless steel columns
0.46.times.5 cm. (Supelco Co., Bellefonte, Pa). The column frits were 2
.mu.m (average pore diameter) titanium encased in a CTFE ring (Upchurch
Scientific, Oak Harbor, Wash.). A Haskell (Burbank, Calif.) DSTV-122
liquid pump was used to provide the drive solvent (water) during the
packing process. The columns were packed at 2000 psi with 50 ml of water
and then 4000 psi with 50 ml of water.
A significant saving in GST analysis time was achieved by performing the
separation on the novel HPLC packing described here. Liver samples were
analyzed in 2 hours, more than ten times faster than similar separations
using Sepharose-6B based affinity supports. Washing and eluting steps were
significantly shorter in duration with the HPLC method. In the analysis of
five human livers (Table 9, below) advantage was taken of the pressure
stability of the HPLC matrix by conducting the washing step at 1.5 ml/min.
Flow rates of at least 2 ml/min can be tolerated in this system with no
loss of affinity bound GST isoenzymes.
The high resolution affinity separation of the GST isoenzymes described
here depended upon the simultaneous application of two concentration
gradients. One gradient consisted of a linearly increasing concentration
of either a glutathione paralog, for instance TER106 (i.e.,
.gamma.E-C(Bz)-.beta.A), or S-butyl glutathione, both of which are known
competitive inhibitors of the GSTs (see Example 9, above). In the loading
step, a GST complexes with an affinity ligand covalently linked to the
stationary phase. As the concentration of the competitive inhibitor
(TER106 or S-butyl glutathione) increases during the elution step, the GST
increasingly partitions into the mobile phase and finally elutes.
The order of elution of a mixture of GSTs is a complex function of their
affinities for both the immobilized affinity ligand and the competitive
inhibitor in the mobile phase. For instance a change in elution order was
observed when comparing the difference in GST elution profiles using two
different eluting ligands. Elution with S-butyl glutathione as compared
with elution with TER106 shifted the Mu class isoenzyme to a broad, longer
retained peak relative to the Alpha class peaks.
In addition, the S-butyl glutathione elution resulted in the coelution of
Ax-y and Ay-y which were resolved with TER106 elution. Twice the
concentration of TER106 to S-butyl glutathione was needed to produce
similar chromatographs, reflecting their differing affinities for the
isoenzymes.
The second gradient was a salt gradient which was applied coincident with
the ligand gradient. Without this gradient the first three peaks coeluted.
Thus this separation depended on both an affinity gradient and a salt
gradient. The salt gradient may be acting to overcome ionic interactions
between the proteins and the stationary phase bound affinity ligand,
S-octyl glutathione, which was expected to have a net negative charge at
pH 6.0.
The recovery of CDNB conjugating activity eluted with an affinity ligand
from the affinity column averaged 76% for three livers while the average
yield of total activity (affinity ligand eluted activity plus unretained
activity) was 83%. The yield of CDNB conjugating activity from the
affinity column described here was comparable to that described in
previous reports using other systems.
The proportion of unretained activity for three livers varied from 3% to
13%. To insure that the fraction of unretained activity was not due to a
system problem such as column saturation, portions of cytosol from the
same liver (L005N) were processed in five consecutive chromatographic
runs. The fraction of unretained activity averaged 2.7% with high and low
values of 3.5% and 2.3%. From this it was concluded that the variation of
unretained activity for different liver samples was a characteristic of
the liver sample.
The identities of GST isoenzymes in the major peaks were identified by
SDS-PAGE electrophoresis, ELISA and reversed-phase HPLC. Liver L006N
appears to contain three forms of Alpha subunits. The reversed-phase
chromatogram of the GSTs isolated from liver L006 shows two peaks which
elute after A1. These later eluting proteins tentatively have been
designated as subunits Ax and Ay, based on further subunit analyses.
For the analysis of human livers, S-butyl glutathione was chosen as the
eluting ligand because it separated Mu class GSTs from the Alpha class. In
addition livers other than L006N had only A1 or in some cases A1 plus only
one of the Ax or Ay subunits so that the complete separation of the Alpha
class dimers afforded by TER106 was not required. The five human livers
were analyzed for GST dimeric content by affinity chromatography and for
monomeric content by reversed-phase chromatography (Table 9).
TABLE 9
______________________________________
Affinity and Reversed-Phase Analysis of Human Liver
______________________________________
Affinity Separation.sup.1
Ax-y
Liver P1-1 Ay-y A1-y A1-x A1-1 Mlb-lb
______________________________________
L006 minor.sup.2
+ + + + minor
L007 minor + + - + -
L004 minor + - + + minor
L005 minor - - minor + +
L002 - - minor
- + minor
______________________________________
Reversed-Phase Separation
P1 M1b A1 Ax Ay
______________________________________
L006 minor minor + + +
L007 minor - + - +
L004 minor minor + + minor
L005 minor minor + minor minor
L002 minor minor + - minor
______________________________________
.sup.1 Affinity Chromatography Buffers; A, 10 mM Sodium Phosphate (pH
6.0); B, 200 mM Sodium Chloride in A; C, 20 mM SButyl Glutathione in B.
Conditions: 0-5 min, A, 0.1 ml/min; 5-21 min, B, 1.5 ml/min; 21-34 min, A
1 ml/min; 34-35 min, A, 0.1 ml/min; 35-155, 0-42% C, 0.1 ml/min.
.sup.2 Symbols: +, Greater than 10% of total peak area; minor, Less than
10% of total peak area; -, Not detected.
The amount of cytosol injected onto the affinity column was equivalent to
approximately 20 mg of liver. In previous reports the predominant forms of
GST in human liver were A1 monomers and A1-1 dimers. This was the case for
the five livers examined here. When Ax or Ay subunits were detected in
appreciable amounts the corresponding heterodimers were also found. For
L007N which had appreciable amounts of monomer Ay, both the homodimer Ay-y
and the heterodimer A1-y as well as A1-1 were detected. Human liver L005N
which had Ax but only a minor amount of Ay exhibited A1-x and A1-1 but no
Ax-x was detected as in the case of L006N. Minor amounts of P1 were
detected by reversed-phase analysis in all five samples and were also
detected by affinity analysis in four of the five livers. Previous
investigations also show Pi to be a minor component in human liver. Small
amounts of the Mu class GSTs were detected by the reversed-phase method.
Due to poor peak shape in affinity chromatography, the detection of the Mu
class is difficult when it constitutes only a minor portion of the total
GST content.
This new affinity chromatography system using HPLC technology has
significant advantages over conventional soft gel affinity systems for the
analysis of the GST dimeric content of tissues. The time of analysis is
reduced by a factor of ten. In addition the sensitivity for this affinity
system is greater because the peak volumes for HPLC affinity are only
about 0.5 milliliter compared to 50-100 milliliters when using
conventional soft gels. This technique also has the advantage of combining
both sample cleanup and analysis into a single step. Other techniques such
as reversed-phase HPLC, chromatofocusing or electrophoresis require a
separate affinity step to batch purify the GSTs before the final analysis.
Affinity chromatography using gradients of a counter ligand in the eluent
is a powerful separation technique. Variation of the biospecific ligand in
the eluent results in chromatographic separations exhibiting different
selectivities. The different selectivities observed are due to the unique
set of binding constants that exist among ligands in their associations
with various isoenzymes. This was the case for the GSTs when comparing
elution profiles using TER106 or S-butyl glutathione as the counter
ligand. Many more compounds of the present invention, as well as other
ligands, interact with GSTs, and any of these could give a unique
separation allowing a custom separation to be developed. In particular,
the analytical methods reported here can be readily scaled up for
preparative isolation of particular GSTs of interest.
EXAMPLE 11
Use of the Compounds of the Invention in Potentiation of Cytotoxic Agents
in Human Cells
This example describes potentiation in human tumor cells of a cytotoxic
agent currently used in cancer chemotherapy by GST inhibitors including
compounds of the present invention, as well as enhanced intracellular
efficacy of esterified forms of these compounds.
HT-29 (human colon adenocarcinoma) cells were obtained from Dr. Roberto
Ceriani (Cancer Research Fund of Contra Costa County, Walnut Creek,
Calif.) and were used in log phase of growth unless otherwise specified.
Chlorambucil (CMB) was obtained from Sigma (St. Louis, Mo.) and was
dissolved in 100% ethanol. All GST inhibitors were dissolved in ethanol,
DMSO, or water just prior to use. The same amount of solvent added to
culture medium served as the vehicle control.
In a modified clonogenic assay for cytotoxicity, cells were suspended at
2.times.10.sup.5 cells/ml in serum-free medium in the presence of vehicle
or inhibitor. Inhibitors were used at concentrations that resulted in
.gtoreq.90% survival when compared to vehicle treated cells. Cells were
incubated for 2 hours, then varying doses of CMB were added. At the end of
a second 2-hour incubation, cells were diluted to 7.5-10.times.10.sup.3 /
ml in serum-containing medium and plated in quadruplicate at 200
.mu.l/well in Microtest III microtiter plates.
Plates were incubated for 6 days and assayed by a modified methylene blue
method. Briefly, cells were fixed with 1.25% glutaraldehyde in PBS then
stained with 0.05% methylene blue in distilled water. Plates were washed
several times in distilled water to remove unretained dye and retained dye
was resolubilized in 0.03N HCl. Plates were read at 650 nm in a Molecular
Devices Vmax plate reader (Molecular Devices, Redwood City, Calif.).
IC.sub.50 values (inhibitor concentration causing 50% reduction in cell
viability) were determined for the drug in the presence or absence of
inhibitor from dose-response curves. A dose modification factor (DMF), a
measure of potentiation of cytotoxicity, was calculated for each inhibitor
by dividing the IC.sub.50 value of CMB without inhibitor treatment by the
IC.sub.50 value for CMB with inhibitor treatment.
Results of potentiation tests with several GST nhibitors in HT29 cell
cultures are summarized in Table 10.
TABLE 10
______________________________________
Potentiation of Chlorambucil Cytotoxicity in Human Cells
by GST Inhibitors and Their Esters
Parent Compound
Diethyl ester
Dose Dose
tested.sup.b tested.sup.b
GST Inhibitor
(No.).sup.a
(.mu.M) DMF.sup.c
(.mu.M)
DMF.sup.c
______________________________________
.gamma.E-C(octyl)-G
(9) N.D. -- 5 0.86 .+-. 0.02
.gamma.E-C(Hx)-.phi.G
(6) 100 1.1 .+-. 0.02
12.5 1.27 .+-. 0.02
.gamma.E-C(Bz)-.phi.G
(4) 100 1.08 .+-. 0.01
12.5 1.65 .+-. 0.04
.gamma.E-C
-- 200 12.5 1.21 .+-. 0.01
(naphthyl)-G
______________________________________
.sup.a Number of compound in Table 5, Example 9, above.
.sup.b Test dose was determined from toxicity curve and analogs were used
at the dose at which .gtoreq.90% survival occurred in the presence of the
analog alone.
.sup.c Dose modification factor. Values are mean .+-. S.D. of 2-3
experiments.
The results in Table 10 show that several GSH analogs found to be
inhibitors of GSH, as shown in Example 9, Table 5, also potentiate killing
of human tumor cells in culture by CMB which is a substrate for various
GSTs. Further, this potentiation is greatly enhanced by esterification
which is designed to enhance uptake of the GST inhibitors. Thus,
.gamma.E-C(Bz)-.phi.G (compound 4, Table 5) at 100 .mu.M was found to
enhance cell killing by CMB, although only slightly, reducing the
concentration CMB needed for 50% cell killing by a DMF of 1.08. In
contrast the dimethyl ester of compound 4 at only 12.5 .mu.M enhanced CMB
cytotoxicity by a factor of 1.65.
Preferential expression of P1-1has been reported in a range of human
tumors. In the present study the efficacy of CME potentiation of the
several GST inhibitors tested correlated directly with their potencies as
inhibitors of the human .pi.-class GST isoenzyme, P1-1, as shown in Table
11.
TABLE 11
______________________________________
Rank Correlation of Chlorambucil Dose Modification Factors of
GST Inhibitors with K.sub.i value for Inhibition of Human GST P1-1
Relative Ki value
Rank DMF.sup.b
Rank
Inhibitor No..sup.a
of parent compound
order
of DEE
order
______________________________________
.gamma.E-C(Bz)-.phi.G
(4) 1 1 1.65 1
.gamma.E-C(Hx)-.phi.G
(6) 2.1 2 1.27 2
.gamma.E-C(naphthyl)-G
-- 3 3 1.21 3
.gamma.E-C(octyl)-G
(9) 4.8 4 .86 4
______________________________________
.sup.a Number of compound in Table 5, Example 9, above.
.sup.b Dose modification factor of diethyl ester. Values are mean .+-.
S.D. of 2-3 experiments.
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